RESUMEN
The re-epithelialization process gets severely dysregulated in chronic nonhealing diabetic foot ulcers/wounds. Keratinocyte growth factor (KGF or FGF-7) is the major modulator of the re-epithelialization process, which regulates the physiological phenotypes of cutaneous keratinocytes. The existing therapeutic strategies of growth factor administration have several limitations. To overcome these, we have designed a KGF-mimetic peptide (KGFp, 13mer) based on the receptor interaction sites in murine KGF. KGFp enhanced migration and transdifferentiation of mouse bone marrow-derived MSCs toward keratinocyte-like cells (KLCs). A significant increase in the expression of skin-specific markers Bnc1 (28.5-fold), Ck5 (14.6-fold), Ck14 (26.1-fold), Ck10 (187.7-fold), and epithelial markers EpCam (23.3-fold) and Cdh1 (64.2-fold) was associated with the activation of ERK1/2 and STAT3 molecular signaling in the KLCs. Further, to enhance the stability of KGFp in the wound microenvironment, it was conjugated to biocompatible 3D porous polymer scaffolds without compromising its active binding sites followed by chemical characterization using Fourier transform infrared spectroscopy, field-emission scanning electron microscopy, dynamic mechanical analysis, and thermogravimetry. In vitro evaluation of the KGFp-conjugated 3D polymer scaffolds revealed its potential for transdifferentiation of MSCs into KLCs. Transplantation of allogeneic MSCGFP using KGFp-conjugated 3D polymer scaffolds in chronic nonhealing type 2 diabetic wounds (db/db transgenic, 50-52 weeks old male mice) significantly enhanced re-epithelialization-mediated wound closure rate (79.3%) as compared to the control groups (Untransplanted -22.4%, MSCGFP-3D polymer scaffold -38.5%). Thus, KGFp-conjugated 3D porous polymer scaffolds drive the fate of the MSCs toward keratinocytes that may serve as potential stem cell delivery platform technology for tissue engineering and transplantation.
Asunto(s)
Factor 7 de Crecimiento de Fibroblastos , Queratinocitos , Andamios del Tejido , Animales , Ratones , Andamios del Tejido/química , Queratinocitos/efectos de los fármacos , Factor 7 de Crecimiento de Fibroblastos/química , Factor 7 de Crecimiento de Fibroblastos/farmacología , Porosidad , Péptidos/química , Péptidos/farmacología , Cicatrización de Heridas/efectos de los fármacos , Piel/efectos de los fármacos , Piel/patología , Polímeros/química , Polímeros/farmacología , Células Madre Mesenquimatosas/efectos de los fármacos , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/metabolismo , Masculino , Regeneración/efectos de los fármacos , Materiales Biomiméticos/química , Materiales Biomiméticos/farmacología , Pie Diabético/tratamiento farmacológico , Pie Diabético/patología , Pie Diabético/terapia , HumanosRESUMEN
Palifermin (Kepivance™) is the first therapeutic approved by the Food and Drug Administration for preventing and managing the oral mucositis provoked by myelotoxic and mucotoxic therapies. Palifermin is a recombinant protein generated from human keratinocyte growth factor (KGF) and imitates the function of endogenous KGF. KGF is an epithelial mitogen involved in various biological processes which belongs to the FGF family. KGF possesses a high level of receptor specificity and plays an important role in tissue repair and maintaining of the mucosal barrier integrity. Based on these unique features, palifermin was developed to enhance the growth of damaged epithelial tissues. Administration of palifermin has shown success in the reduction of toxicities of chemotherapy and radiotherapy, and improvement of the patient's quality of life. Notwithstanding all merits, the clinical application of palifermin is limited owing to its instability and production challenges. Hence, a growing number of ongoing researches are designed to deal with these problems and enhance the physicochemical and pharmaceutical properties of palifermin. In the current review, we discuss KGF structure and function, potential therapeutic applications of palifermin, as well as the latest progress in the production of recombinant human KGF and its challenges ahead.
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Factor 7 de Crecimiento de Fibroblastos/química , Factor 7 de Crecimiento de Fibroblastos/farmacología , Factor 7 de Crecimiento de Fibroblastos/uso terapéutico , Estomatitis/tratamiento farmacológico , Antineoplásicos/uso terapéutico , Apoptosis/efectos de los fármacos , Proteínas Portadoras , Movimiento Celular/efectos de los fármacos , Factor 7 de Crecimiento de Fibroblastos/genética , Heparina , Humanos , Modelos Moleculares , Conformación Proteica , Calidad de Vida , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos , Estomatitis/prevención & control , Cicatrización de Heridas/efectos de los fármacosRESUMEN
BACKGROUND: Recombinant Keratinocyte Growth Factor (rHuKGF) is a therapeutic protein used widely in oral mucositis after chemotherapy in various cancers, stimulating lung morphogenesis and gastrointestinal tract cell proliferation. In this research study, chitosan-rHuKGF polymeric complex was implemented to improve the stability of rHuKGF and used as rejuvenation therapy for the treatment of oral mucositis in cancer patients. OBJECTIVE: Complexation of rHuKGF with mucoadhesive low molecular weight chitosan to protect rHuKGF from proteolysis and investigate the effect of chitosan-rHuKGF complex on the proliferation rate of FHs 74 Int cells. METHODS: The interaction between chitosan and rHuKGF was studied by molecular docking. Malvern ZetaSizer Nano Zs and Fourier-Transform Infrared spectroscopy (FTIR) tests were carried out to characterize the chitosan-rHuKGF complex. In addition, SDS-PAGE was performed to investigate the interaction between chitosan-rHuKGF complex and pepsin. The effect of chitosan-rHuKGF complex on the proliferation rate of FHs 74 Int cells was studied by MTT assay. RESULTS: Chitosan-rHuKGF complex was formed through the hydrogen bonding proven by the docking studies. A stable chitosan-rHuKGF complex was formed at pH 4.5 and was protected from proteolysis and assessed by SDS PAGE. According to the MTT assay results, chitosan-rHuKGF complex increased the cell proliferation rate of FHs 74 Int cells. CONCLUSION: The developed complex improved the stability and the biological function of rHuKGF.
Asunto(s)
Adhesivos/química , Proliferación Celular/efectos de los fármacos , Quitosano/química , Factor 7 de Crecimiento de Fibroblastos/química , Proteolisis/efectos de los fármacos , Adhesivos/metabolismo , Adhesivos/farmacología , Proliferación Celular/fisiología , Células Cultivadas , Quitosano/metabolismo , Quitosano/farmacología , Feto , Factor 7 de Crecimiento de Fibroblastos/metabolismo , Humanos , Mucosa Intestinal/citología , Mucosa Intestinal/efectos de los fármacos , Mucosa Intestinal/metabolismo , Simulación del Acoplamiento Molecular/métodos , Estructura Secundaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Espectroscopía Infrarroja por Transformada de Fourier/métodosRESUMEN
Aim: Diabetic wound healing is seriously interrupted, and administration of KGF for wound treatment is restricted by its inherent instability. We aim to develop an ideal way toward KGF stabilization, thus improving diabetic wound healing. Materials & methods: We conjugated KGF with gold nanoparticles (GNPs) and determined the stability and binding affinity. Biological effects of conjugates (KGF-GNPs) were evaluated in vitro and in an animal model. Results: KGF-GNPs revealed high stability under hostile circumstances because of the preserved secondary structure and possessed elevated binding affinity to KGF receptor. Moreover, application of KGF-GNPs contributed to accelerated wound recovery in diabetic rats, including re-epithelialization and contraction. Conclusion: KGF-GNPs were promising for future clinical application for diabetic wound therapy.
Asunto(s)
Factor 7 de Crecimiento de Fibroblastos/química , Factor 7 de Crecimiento de Fibroblastos/uso terapéutico , Oro/química , Nanopartículas del Metal/química , Factor de Crecimiento Transformador beta1/química , Cicatrización de Heridas/efectos de los fármacos , Animales , Western Blotting , Línea Celular , Línea Celular Tumoral , Dicroismo Circular , Femenino , Humanos , Cinética , Estructura Secundaria de Proteína , Ratas , Resonancia por Plasmón de SuperficieRESUMEN
The need for direct biomaterial-based delivery of growth factors to wound surfaces to aid in wound healing emphasizes the importance of interfacial interactions between the biomaterial and the wound surface. These interactions include the spatial localization of growth factor, the surface intensity of growth factor in contact with the wound, and the release profile of growth factor to the wound surface. The authors report the use of time-of-flight secondary ion mass spectrometry to determine the relationship between biomaterial surface chemistry and the spatial localization of growth factor. They have implemented a novel application of total internal reflectance fluorescence (TIRF) microscopy to measure the surface intensity and release of growth factor in contact with a glass substrate that has been used to model a wound surface. Detailed information regarding TIRF experiments has been included to aid in future studies regarding the biomaterial delivery to interfaces. The authors have evaluated the effects of (hydroxyethyl)methacrylate (HEMA) homopolymer, 5.89% methyl methacrylate/HEMA, and 5.89% methacrylic acid/HEMA surface chemistry on the spatial localization of AlexaFluor 488-labeled keratinocyte growth factor (AF488-KGF), AF488-KGF surface intensity at the copolymer surface, and release to a glass substrate. KGF is known to promote re-epithelialization in wound healing. The results show that the two copolymers allow for increased surface coverage, surface intensity, and release of AF488-KGF in comparison to the homopolymer. It is likely that differences in these three aspects could have a profound effect on the wound healing response.
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Factor 7 de Crecimiento de Fibroblastos/química , Colorantes Fluorescentes/química , Queratinocitos/metabolismo , Espectrometría de Masas , Metacrilatos/química , Coloración y Etiquetado , Humanos , Queratinocitos/citología , Microscopía Fluorescente , Propiedades de SuperficieRESUMEN
Regenerative therapy with keratinocyte growth factor (KGF) is a novel therapeutic approach for treatment of chronic wounds. However, KGF cannot be used directly to the wound site due to its physicochemical instability. In previous study, sacran, a natural megamolecular polysaccharide, showed potential properties as a biomaterial for hydrogel film in wound healing. In this study, we fabricated sacran hydrogel film containing KGF (Sac/KGF-HF) and evaluated the effects of Sac/KGF-HF on fibroblasts migration and re-epithelialization process. We successfully prepared a homogenous and -amorphous Sac/KGF-HF by a casting method. In addition, Sac/KGF-HF had a high swelling ratio and flexibility. Sac/KGF-HF promoted a migration process of NIH3T3 cells and improved wound healing ability in mice with a percentage of wound closure reaching 90.4% at 9 d. Interestingly, the addition of KGF in Sac-HF considerably increased the number of epithelial cells compared to control, which is important in the re-epithelialization process. It could be concluded that KGF in Sac-HF has the potential for promoting Sac-HF abilities in wound healing process.
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Factor 7 de Crecimiento de Fibroblastos/metabolismo , Fibroblastos/efectos de los fármacos , Metilgalactósidos/farmacología , Polisacáridos/farmacología , Cicatrización de Heridas/efectos de los fármacos , Animales , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Factor 7 de Crecimiento de Fibroblastos/química , Metilgalactósidos/química , Ratones , Ratones Endogámicos BALB C , Células 3T3 NIH , Polisacáridos/químicaRESUMEN
Keratinocyte growth factor (KGF) has a good therapeutic effect on injured corneas. However, due to the washout of tears and blinking, locally administrated KGF usually has a short residence time on the surface of an injured cornea, resulting in its poor bioavailability. Herein, a bioadhesive hydrogel is described produced using cysteine-modified γ-polyglutamic acid (PGA-Cys) as the hydrogel-forming material to locally deliver KGF. A series of PGA-Cys polymers with different graft ratios of cysteine were firstly synthesized and carefully characterized. Thereafter, the PGA-Cys hydrogel was screened by changing the graft ratio of cysteine and polymer concentration, and the apparent viscosities and bioadhesive force were also carefully investigated. It was found that PGA-Cys polymers with different graft ratios of cysteine exhibited tunable apparent viscosity and bioadhesive properties at the same polymer concentration. When PGA-Cys with a graft ratio of 1.5 mmol g-1 of cysteine (PGA-Cys-1.5) was used as hydrogel-forming material, the hydrogel exhibited a good gelation property with an apparent viscosity of 5.2 Pa s and strong bioadhesive force of 167 ± 0.5 mN. In vitro release study showed that KGF was slowly released from PGA-Cys-1.5 hydrogel over a longer time in comparison to PGA solution alone. Moreover, PGA-Cys-1.5 hydrogel enabled most of the encapsulated KGF to be retained on the cornea and conjunctiva after local administration. Meanwhile, the morphology of the corneal epithelium in the alkali-injured cornea of mice was well repaired after 7 days of treatment with KGF-PGA-Cys-1.5 hydrogel. The therapeutic mechanism was strongly associated with inhibiting corneal inflammation and neovascularization, promoting proliferation of the corneal epithelium and inhibiting apoptosis. Overall, the use of the bioadhesive PGA-Cys hydrogel with a suitable KGF release profile may be a more promising approach than using PGA solution alone and KGF to repair injured corneas.
Asunto(s)
Lesiones de la Cornea/tratamiento farmacológico , Portadores de Fármacos/química , Factor 7 de Crecimiento de Fibroblastos/química , Hidrogeles/química , Ácido Poliglutámico/análogos & derivados , Adhesividad , Animales , Apoptosis/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Lesiones de la Cornea/patología , Cisteína/química , Preparaciones de Acción Retardada , Factor 7 de Crecimiento de Fibroblastos/farmacología , Factor 7 de Crecimiento de Fibroblastos/uso terapéutico , Fibrosis , Ratones , Células 3T3 NIH , Neovascularización Patológica/tratamiento farmacológico , Ácido Poliglutámico/químicaRESUMEN
BACKGROUND: The recombinant human truncated Keratinocyte growth factor (Palifermin) is the only FDA approved medicine for the treatment of oral mucositis. The Keratinocyte growth factor is a fairly unstable protein due to its high aggregation propensity and therefore its expression as a secretory protein may results in the production of a protein with more stability, higher solubility, better folding, enhanced biological activity, N-terminal authenticity and simplified downstream processing. OBJECTIVE: The aim of this study was in silico evaluation of 31 different secretory signal peptides to determine the best theoretical candidates for the secretory production of recombinant truncated human KGF in E. coli. METHODS: Thirty different prokaryotic signal peptides experimentally shown to be capable of recombinant protein secretion in E.coli, along with the native KGF signal peptide were selected for further investigations. The signal peptide sequences were retrieved from the UniProt database. The ability of SPs to act as a secretory leader peptide for rhKGF and the location of cleavage sites were predicted by SignalP 4.1. Physicochemical properties of the signal peptides, which may influence protein secretion, were analyzed by ProtParam and PROSOII. Furthermore, the mRNA secondary structure and Gibbs free energy profile of the selected SPs were analyzed in the fusion state with the rhKGF using Visual Gene Developer package. RESULTS AND CONCLUSION: Computational analysis of the physicochemical properties affecting protein secretion identified Sec-B dependent OmpC, Bla, and StaI and SRP dependent TolB signal peptides as the best theoretical candidates for the secretory production of recombinant truncated human KGF in E.coli.
Asunto(s)
Simulación por Computador , Factor 7 de Crecimiento de Fibroblastos/química , Señales de Clasificación de Proteína , Proteínas Recombinantes/química , Secuencia de Aminoácidos , Escherichia coli/química , Factor 7 de Crecimiento de Fibroblastos/genética , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , ARN Mensajero/química , Proteínas Recombinantes/genética , Solubilidad , TermodinámicaRESUMEN
Expediting the wound-healing process is critical for patients chronically ill from nonhealing wounds and diseases such as hemophilia or diabetes or who have suffered trauma including easily infected open wounds. FDA-approved external tissue sealants include the topical application of fibrin gels, which can be 500 times denser than natural fibrin clots. With lower clot porosity and higher polymerization rates than physiologically formed fibrin clots, the commercial gels quickly stop blood loss but impede the later clot degradation kinetics and thus retard tissue-healing rates. The fibrin nanoparticles (FBNs) described here are constructed from physiologically relevant fibrin concentrations that support new tissue and dermal wound scaffold formation when coupled with growth factors. The FBNs, synthesized in a microfluidic droplet generator, support cell adhesion and traction generation, and when coupled to keratinocyte growth factor (KGF), support cell migration and in vivo wound healing. The FBN-KGF particles enhance cell migration in vitro greater than FBN alone or free KGF and also improve healing outcomes in a murine full thickness injury model compared to saline, bulk fibrin sealant, free KGF, or bulk fibrin mixed with KGF treatments. Furthermore, FBN can be potentially administered with other tissue-healing factors and inflammatory mediators to improve wound-healing outcomes.
Asunto(s)
Fibrina/química , Factor 7 de Crecimiento de Fibroblastos/química , Factor 7 de Crecimiento de Fibroblastos/uso terapéutico , Nanopartículas/química , Cicatrización de Heridas/efectos de los fármacos , Animales , Materiales Biocompatibles/química , Movimiento Celular/efectos de los fármacos , Ratones , MicrofluídicaRESUMEN
The development of hydrogels for protein delivery requires protein-hydrogel interactions that cause minimal disruption of the protein's biological activity. Biological activity can be influenced by factors such as orientational accessibility for receptor binding and conformational changes, and these factors can be influenced by the hydrogel surface chemistry. (Hydroxyethyl)methacrylate (HEMA) hydrogels are of interest as drug delivery vehicles for keratinocyte growth factor (KGF) which is known to promote re-epithelialization in wound healing. The authors report here the surface characterization of three different HEMA hydrogel copolymers and their effects on the orientation and conformation of surface-bound KGF. In this work, they characterize two copolymers in addition to HEMA alone and report how protein orientation and conformation is affected. The first copolymer incorporates methyl methacrylate (MMA), which is known to promote the adsorption of protein to its surface due to its hydrophobicity. The second copolymer incorporates methacrylic acid (MAA), which is known to promote the diffusion of protein into its surface due to its hydrophilicity. They find that KGF at the surface of the HEMA/MMA copolymer appears to be more orientationally accessible and conformationally active than KGF at the surface of the HEMA/MAA copolymer. They also report that KGF at the surface of the HEMA/MAA copolymer becomes conformationally unfolded, likely due to hydrogen bonding. KGF at the surface of these copolymers can be differentiated by Fourier-transform infrared-attenuated total reflectance spectroscopy and time-of-flight secondary ion mass spectrometry in conjunction with principal component analysis. The differences in KGF orientation and conformation between these copolymers may result in different biological responses in future cell-based experiments.
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Portadores de Fármacos , Factor 7 de Crecimiento de Fibroblastos/química , Hidrogeles , Proteínas Inmovilizadas/química , Metacrilatos , Conformación Proteica , Espectrometría de Masa de Ion Secundario , Espectroscopía Infrarroja por Transformada de FourierRESUMEN
Keratinocyte growth factor (KGF) has been demonstrated to specifically stimulate the multiplication and migration of keratinocytes. However, due to rapid degradation, the results of topical application of growth factors on wounds are unsatisfactory. In this study, we cross-linked KGF to the surface of gold nanoparticles (GNPs) and explored their effects on wound healing. The as-synthesized nanocomposite (KGF-GNPs) displayed good colloidal stability, decent biocompatibility as well as negligible cellular cytotoxicity. The in vitro cellular experimental results demonstrated that KGF-GNPs could effectively promote the proliferation of keratinocytes in contrast to bare GNPs or KGF. Furthermore, in animal full-thickness wound model, KGF-GNPs are more conducive to wound healing than bare GNPs or KGF. KGF-GNPs enhanced wound healing by promoting wound re-epithelialization rather than granulation. The superior biocompatibility, colloidal depressiveness and biological activity of this nanocomposite indicate that it could be utilized as a promising wound healing drug for clinical application in the future.
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Factor 7 de Crecimiento de Fibroblastos/administración & dosificación , Oro/química , Queratinocitos/citología , Nanopartículas del Metal/administración & dosificación , Cicatrización de Heridas , Administración Tópica , Animales , Células Cultivadas , Femenino , Factor 7 de Crecimiento de Fibroblastos/química , Queratinocitos/efectos de los fármacos , Nanopartículas del Metal/química , Ratas , Ratas Sprague-DawleyRESUMEN
The drug development of FGF7 has been restricted by its toxicity to the host, low expression, poor stability, and easy degradation. Recent studies have shown that Halo-tag-flanked recombinant human FGF7 can solve the problem of toxicity; however, its biological activity is unknown. This study aimed to explore the activity of Halo-rhFGF7 and rhFGF7 on acute liver injury in vitro and in vivo. The rhFGF7 is expressed with a N-terminal Halo-tag, followed by a tobacco etch virus (TEV) protease cleavage site, in Escherichia coli BL21 (DE3) pLysS in this study. The products could stimulate the proliferation of carbon tetrachloride-damaged L-O2 cells (normal human liver cells); they also inhibited cell apoptosis. Due to the use of the Halo, the protein could be tracked using fluorescence localization. Recombinant protein exerted a protective effect on the acute liver injury model in vitro and in vivo. The MTT assay and Western blot analysis showed that this protective effect is realized through various paths, including promoting proliferation, inhibiting cell apoptosis and anti-inflammatory. In conclusion, Halo-rhFGF7 and rhFGF7 displayed an excellent protective effect on acute liver injury. The present study provided an experimental basis and data support for further research on rhFGF7.
Asunto(s)
Enfermedad Hepática Inducida por Sustancias y Drogas/metabolismo , Factor 7 de Crecimiento de Fibroblastos/farmacología , Hígado/efectos de los fármacos , Proteínas Recombinantes de Fusión/farmacología , Animales , Apoptosis/efectos de los fármacos , Tetracloruro de Carbono/toxicidad , Línea Celular , Proliferación Celular/efectos de los fármacos , Enfermedad Hepática Inducida por Sustancias y Drogas/patología , Escherichia coli/genética , Factor 7 de Crecimiento de Fibroblastos/química , Factor 7 de Crecimiento de Fibroblastos/genética , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Humanos , Hígado/citología , Ratones , Sustancias Protectoras/química , Sustancias Protectoras/farmacología , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genéticaRESUMEN
Keratinocyte growth factor (KGF), a member of the fibroblast growth factor (FGF) family, has been implicated in some biological processes such as cell proliferation, development and differentiation. High mitogenic activity of this protein has made it very suitable for repairing radiation-and chemotherapy-induced damages. Palifermin, which has been developed from human KGF, is clinically applied to reduce the incidence and duration of cancer therapeutic agents. However, the activity of Palifermin is limited during treatment due to its poor stability. In this study, we have improved the stability and activity of recombinant human KGF (Palifermin) using a computational mutagenesis approach. According to the KGF multiple sequence alignment among different species as well as literature-based information, we have generated several mutations using PyMOL program and evaluated their effects on the stability and activity of KGF in silico. In order to preserve the KGF activity, we did not change the predicted functional residues. Prior to mutagenesis, the 3D structure of rhKGF was predicted by Modeller v9.15 program and quantitative evaluation of predicted models were carried out using VADAR and PROSESS servers. The stability and activity of rhKGF mutants were analyzed using GROMACS molecular dynamics (MD) simulations and docking tools, respectively. The results showed that N159S (N105S in rhKGF sequence) and I172V (I118V in rhKGF) substitutions caused an increased stability and affinity of the rhKGF to Fibroblast growth factor receptor 2 (FGFR2). We will evaluate the effects of favorable mutations on the rhKGF stability and activity in vitro.
Asunto(s)
Sustitución de Aminoácidos , Factor 7 de Crecimiento de Fibroblastos/química , Factor 7 de Crecimiento de Fibroblastos/genética , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Análisis de Secuencia de Proteína/métodos , Programas Informáticos , Animales , Bovinos , Factor 7 de Crecimiento de Fibroblastos/metabolismo , Factor 7 de Crecimiento de Fibroblastos/uso terapéutico , Humanos , Ratones , Mutagénesis , Mutación Missense , Neoplasias/tratamiento farmacológico , Dominios Proteicos , Estabilidad Proteica , Ratas , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos/química , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos/genética , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos/metabolismo , Ovinos , Relación Estructura-Actividad , PorcinosRESUMEN
Molecular interactions between mesenchymal-derived Keratinocyte growth factor (KGF) and Kit ligand (KITLG) are essential for follicular development. These factors are expressed by theca and granulosa cells. We determined full length coding sequence of buffalo KGF and KITLG proteins having 194 and 274 amino acids, respectively. The recombinant KGF and KITLG proteins were solubilized in 10 mM Tris, pH 7.5 and 50 mM Tris, pH 7.4 and purified using Ni-NTA column and GST affinity chromatography, respectively. The purity and molecular weight of His-KGF (~23 kDa) and GST-KITLG (~57 kDa) proteins were confirmed by SDS-PAGE and western blotting. The co-immunoprecipitation assay accompanied with computational analysis demonstrated the interaction between KGF and KITLG proteins. We deduced 3D structures of the candidate proteins and assessed their binding based on protein docking. In the process, KGF specific residues, Lys123, Glu135, Lys140, Lys155 and Trp156 and KITLG specific ones, Ser226, Phe233, Gly234, Ala235, Phe236, Trp238 and Lys239 involved in the formation of KGF-KITLG complex were detected. The hydrophobic interactions surrounding KGF-KITLG complex affirmed their binding affinity and stability to the interacting interface. Additionally, in-silico site directed mutagenesis enabled the assessment of changes that occurred in the binding energies of mutated KGF-KITLG protein complex. Our results demonstrate that in the presence of KITLG, KGF mimics its native binding mode suggesting all the KGF residues are specific to their binding complex. This study provides an insight on the critical amino acid residues participating in buffalo ovarian folliculogenesis.
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Búfalos/metabolismo , Factor 7 de Crecimiento de Fibroblastos/metabolismo , Folículo Ovárico/metabolismo , Factor de Células Madre/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Análisis por Conglomerados , Femenino , Factor 7 de Crecimiento de Fibroblastos/química , Factor 7 de Crecimiento de Fibroblastos/genética , Interacciones Hidrofóbicas e Hidrofílicas , Simulación del Acoplamiento Molecular , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Unión Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Factor de Células Madre/química , Factor de Células Madre/genéticaRESUMEN
Three forms of recombinant human keratinocyte growth factor 1 (rhKGF1) with or without the native signal peptide or a 23-amino acid truncation were expressed in Spodoptera frugiperda 9 (Sf9) cells by designing with insect codon usage. Immunoblotting demonstrated that these rhKGF1 proteins were recognized by a human anti-KGF1 antibody. The multiplicity of infection and timing of harvest had a significant effect on protein yield, protein quality, and cytotoxicity. Our results indicated that the native signal peptide directed KGF1 secretion from insect cells, reaching a maximum at 60 h postinfection. Although secretion of rhKGF1194 was less efficient than that of rhKGF1163 and rhKGF1140, protein secretion is an attractive pathway for simple purification of biologically active rhKGF1 at a high yield. Moreover, the sizes of rhKGF1194 and rhKGF1163 were similar (20 kDa), suggesting that the signal peptide may be recognized and removed in Sf9 cells. A 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide assay was used to analyze the biological function of rhKGF1, indicating that the three forms of rhKGF1 had a similar mitogenic function in BaF3 cells. Furthermore, to elucidate the effect of rhKGF1 on cytoprotection of liver cells, we used KGF1 pretreatment of an acute liver injury model. The results indicated that rhKGF1 prevented necrosis and apoptosis of CCl4-treated HL7702 cells in vitro and in vivo. These results suggest that KGF1 may be a candidate therapeutic drug for acute liver injury.
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Factor 7 de Crecimiento de Fibroblastos/biosíntesis , Expresión Génica , Hepatocitos/efectos de los fármacos , Hepatocitos/fisiología , Animales , Línea Celular , Citoprotección , Factor 7 de Crecimiento de Fibroblastos/química , Factor 7 de Crecimiento de Fibroblastos/genética , Factor 7 de Crecimiento de Fibroblastos/metabolismo , Humanos , Datos de Secuencia Molecular , Peso Molecular , Señales de Clasificación de Proteína , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Análisis de Secuencia de ADN , SpodopteraRESUMEN
Keratinocyte growth factor 1 (KGF-1) has proven useful in the treatment of pathologies associated with dermal adnexae, liver, lung, and the gastrointestinal tract diseases. However, poor stability and short plasma half-life of the protein have restricted its therapeutic applications. While it is possible to improve the stability and extend the circulating half-life of recombinant human KGF-1 (rhKGF-1) using solution-phase PEGylation, such preparations have heterogeneous structures and often low specific activities due to multiple and/or uncontrolled PEGylation. In the present study, a novel solid-phase PEGylation strategy was employed to produce homogenous mono-PEGylated rhKGF-1. RhKGF-1 protein was immobilized on a Heparin-Sepharose column and then a site-selective PEGylation reaction was carried out by a reductive alkylation at the N-terminal amino acid of the protein. The mono-PEGylated rhKGF-1, which accounted for over 40% of the total rhKGF-1 used in the PEGylation reaction, was purified to homogeneity by SP Sepharose ion-exchange chromatography. Our biophysical and biochemical studies demonstrated that the solid-phase PEGylation significantly enhanced the in vitro and in vivo biostability without affecting the over all structure of the protein. Furthermore, pharmacokinetic analysis showed that modified rhKGF-1 had considerably longer plasma half-life than its intact counterpart. Our cell-based analysis showed that, similar to rhKGF-1, PEGylated rhKGF-1 induced proliferation in NIH 3T3 cells through the activation of MAPK/Erk pathway. Notably, PEGylated rhKGF-1 exhibited a greater hepatoprotection against CCl(4)-induced injury in rats compared to rhKGF-1.
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Factor 7 de Crecimiento de Fibroblastos/química , Factor 7 de Crecimiento de Fibroblastos/farmacología , Polietilenglicoles/química , Proteínas Recombinantes/química , Proteínas Recombinantes/farmacología , Aldehídos/química , Animales , Sitios de Unión , Tetracloruro de Carbono/efectos adversos , Enfermedad Hepática Inducida por Sustancias y Drogas/prevención & control , Citoprotección/efectos de los fármacos , Factor 7 de Crecimiento de Fibroblastos/farmacocinética , Humanos , Fallo Hepático Agudo/inducido químicamente , Fallo Hepático Agudo/prevención & control , Masculino , Fragmentos de Péptidos/química , Estabilidad Proteica , Ratas , Proteínas Recombinantes/farmacocinética , Reproducibilidad de los Resultados , Relación Estructura-Actividad , Especificidad por SustratoRESUMEN
Chronic wounds are associated with poor epidermal and dermal remodeling. Previous work has shown the efficacy of keratinocyte growth factor (KGF) in reepithelialization and elastin in dermal wound healing. Here we demonstrate the fabrication of a fusion protein comprising of elastin-like peptides and KGF. This fusion protein retains the performance characteristics of KGF and elastin as evidenced by its enhancement of keratinocyte and fibroblast proliferation. It also preserved the characteristic elastin-like peptides inverse phase transitioning allowing the recombinant protein to be expressed in bacterial hosts (such as Escherichia coli) and purified rapidly and easily using inverse temperature cycling. The fusion protein self-assembled into nanoparticles at physiological temperatures. When applied to full thickness, wounds in Lepr(db) diabetic mice these particles enhanced reepithelialization and granulation, by 2- and 3-fold respectively, when compared to the controls. The data strongly suggests that these self-assembled nanoparticles may be beneficial in the treatment of chronic wounds resulting from diabetes or other underlying circulatory conditions.
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Elastina/química , Factor 7 de Crecimiento de Fibroblastos/química , Complejos Multiproteicos/uso terapéutico , Nanopartículas/uso terapéutico , Cicatrización de Heridas/efectos de los fármacos , Heridas y Lesiones/tratamiento farmacológico , Análisis de Varianza , Animales , Western Blotting , Masculino , Ratones , Microscopía Electrónica de Transmisión , Complejos Multiproteicos/química , Complejos Multiproteicos/farmacología , Nanopartículas/química , Nanopartículas/ultraestructuraAsunto(s)
Cardiología , Úlcera Varicosa/terapia , Antibacterianos/farmacología , Antiinfecciosos Locales , Vendajes , Plaquetas/metabolismo , Coloides/química , Vendajes de Compresión , Factor 7 de Crecimiento de Fibroblastos/química , Miel , Humanos , Placebos , Ensayos Clínicos Controlados Aleatorios como Asunto , Piel/metabolismo , Cicatrización de HeridasRESUMEN
BACKGROUND: Keratinocyte growth factor (KGF) significantly influences epithelial wound healing. The aim of this study was to isolate KGF phage model peptides from a phage display 7-mer peptide library to evaluate their effect on promoting epidermal cell proliferation. METHODS: A phage display 7-mer peptide library was screened using monoclonal anti-human KGF antibody as the target. Enzyme linked immunosorbent assay (ELISA) was performed to select monoclonal phages with good binding activity. DNA sequencing was done to find the similarities of model peptides. Three-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay, immunofluorescence assay and quantitative real-time PCR analysis were employed to evaluate the effect of the phage model peptides on epidermal cells. RESULTS: Thirty-three out of fifty-eight (56.9%) of the isolated monoclonal phages exhibited high binding activity by ELISA. Ten of fifteen obtained phage model peptides were similar to KGF or epidermal growth factor (EGF). MTT assay data showed that four (No. 1 - 4) of the ten phage model peptides could promote epidermal cell proliferation. The expression of keratinocyte growth factor receptor (KGFR) mRNA in the KGF control group and the two phage model peptide groups (No. 1 and No. 2) increased. Expression of c-Fos mRNA and c-Jun mRNA in the KGF control group increased, but did not increase in the four phage model peptide groups (No.1 - 4). CONCLUSION: Four phage model peptides isolated from the phage display 7-mer peptide library can safely promote epidermal cell proliferation without tumorigenic effect.
Asunto(s)
Proliferación Celular/efectos de los fármacos , Células Epidérmicas , Factor 7 de Crecimiento de Fibroblastos/química , Factor 7 de Crecimiento de Fibroblastos/farmacología , Péptidos/química , Péptidos/farmacología , Células Cultivadas , Ensayo de Inmunoadsorción Enzimática , Humanos , Biblioteca de Péptidos , Reacción en Cadena de la Polimerasa , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos/genéticaRESUMEN
As part of a study of elicited angiogenesis, hyaluronan (HA)-based hydrogels crosslinked by polyethylene glycol diacrylate (PEGDA) were loaded with combinations of the cytokine growth factors vascular endothelial growth factor (VEGF), angiopoietin-1 (Ang-1), keratinocyte growth factor (KGF) and platelet-derived growth factor (PDGF). GF release in vivo was controlled by covalent incorporation of thiol-modified heparin into thiolated HA hydrogels, which were injected into the ear pinnae of mice and allowed to crosslink in situ. GF release in vivo was controlled by covalent incorporation of thiol-modified heparin in the gels. The ears were harvested at 7 or 14 days post-implantation, and vascularization evaluated via a Neovascularization Index (NI). The study demonstrates that in situ gelling implants produced no gross inflammation, redness or swelling, and an improved tolerance compared to HA-based dry film implants. All treatments showed significantly more vascularization than either contralateral ears or ears receiving a sham surgery. The maximum response was observed after 14 days in the ears receiving 0.3% Hp, gelatin-containing gels loaded with VEGF + KGF (NI = 3.91). The study revealed injected growth factor-loaded HA-based hydrogels can successfully produce localized controllable vascularization, while minimizing tissue necrosis, polymorphonuclear leukocytes and inflammation. The ability to target and controllably release growth factors can prove a useful tool in specific diseased tissue/organ angiogenesis.