RESUMEN
Peptides are gaining substantial attention as therapeutics for human diseases. However, they have limitations such as low bioavailability and poor pharmacokinetics. Periostin, a matricellular protein, can stimulate the repair of ischemic tissues by promoting angiogenesis. We have previously reported that a novel angiogenic peptide (amino acids 142-151) is responsible for the pro-angiogenic activity of periostin. To improve the in vivo delivery efficiency of periostin peptide (PP), we used proteins self-assembled into a hollow cage-like structure as a drug delivery nanoplatform in the present study. The periostin peptide was genetically inserted into lumazine synthase (isolated from Aquifex aeolicus) consisting of 60 identical subunits with an icosahedral capsid architecture. The periostin peptide-bearing lumazine synthase protein cage nanoparticle with 60 periostin peptides multivalently displayed was expressed in Escherichia coli and purified to homogeneity. Next, we examined angiogenic activities of this periostin peptide-bearing lumazine synthase protein cage nanoparticle. AaLS-periostin peptide (AaLS-PP), but not AaLS, promoted migration, proliferation, and tube formation of human endothelial colony-forming cells in vitro. Intramuscular injection of PP and AaLS-PP increased blood perfusion and attenuated severe limb loss in the ischemic hindlimb. However, AaLS did not increase blood perfusion or alleviate tissue necrosis. Moreover, in vivo administration of AaLS-PP, but not AaLS, stimulated angiogenesis in the ischemic hindlimb. These results suggest that AaLS is a highly useful nanoplatform for delivering pro-angiogenic peptides such as PP. [BMB Reports 2022; 55(4): 175-180].
Asunto(s)
Nanopartículas , Neovascularización Patológica , Animales , Miembro Posterior , Humanos , Isquemia/tratamiento farmacológico , Isquemia/metabolismo , Neovascularización Patológica/tratamiento farmacológico , Neovascularización Fisiológica , Péptidos/farmacologíaRESUMEN
Angiogenic peptides have therapeutic potential for the treatment of chronic ischemic diseases. Periostin, an extracellular matrix protein expressed in injured tissues, promotes angiogenesis and tissue repair. We previously reported that in vivo administration of both recombinant full-length protein and the first FAS I domain of periostin alleviated peripheral artery occlusive disease by stimulating the migration of humane endothelial colony forming cells (ECFCs) and subsequent angiogenesis. In the present study, we ascertained the peptide sequence responsible for the periostin-induced angiogenesis. By serial deletion mapping of the first FAS I domain, we identified a peptide sequence (amino acids 142-151) of periostin for stimulation of chemotactic migration, adhesion, proliferation and endothelial tube formation of human ECFCs in vitro. Chemotactic migration of ECFCs induced by the periostin peptide was blocked by pre-incubation with an anti-ß5 integrin neutralizing antibody. Treatment of ECFCs with the periostin peptide led to phosphorylation of both AKT and ERK, and pretreatment of ECFCs with the MEK-ERK pathway inhibitor U0126 or the PI3K-AKT pathway inhibitors, LY294002 or Wortmannin, blocked the periostin peptide-stimulated migration of ECFCs. These results suggest that the synthetic periostin peptide can be applied for stimulating angiogenic and therapeutic potentials of ECFCs.
Asunto(s)
Proteínas Angiogénicas/metabolismo , Moléculas de Adhesión Celular/química , Androstadienos/farmacología , Anticuerpos Neutralizantes/inmunología , Butadienos/farmacología , Moléculas de Adhesión Celular/inmunología , Moléculas de Adhesión Celular/metabolismo , Células Cultivadas , Cromonas/farmacología , Células Progenitoras Endoteliales/citología , Células Progenitoras Endoteliales/efectos de los fármacos , Células Progenitoras Endoteliales/metabolismo , Humanos , Morfolinas/farmacología , Neovascularización Patológica , Nitrilos/farmacología , Fosforilación , WortmaninaRESUMEN
Periostin (POSTN), a secreted homodimeric protein that binds integrins αvß3, αvß5, and α6ß4, was originally found to be expressed in fetal tissues and in the adult upon injury particularly bone fractures due to its role in remodelling and repair. Recently it was found to be over-expressed in human breast cancer and a variety of other tumour types including head and neck squamous cell carcinoma, where its overexpression correlates with increased tumour invasion. Progress in studying its functional role in tumour pathogenesis has been hampered by the paucity of antibodies for its specific and sensitive detection. It has proven very difficult to obtain monoclonal antibodies (mAbs) against this highly conserved protein but we report here that combining infection of mice with lactate dehydrogenase elevating virus (LDV), a B cell activating arterivirus, with conjugation of human POSTN to ovalbumin as an immunogenic carrier, enabled us to develop six mAbs recognizing both human and mouse POSTN and inhibiting its binding to αvß3 integrin. Two of the mAbs, MPB4B1 and MPC5B4, were tested and found to inhibit POSTN-induced migration of human endothelial colony forming cells. All six mAbs recognized amino acids 136-51 (APSNEAWDNLDSDIRR) within the POSTN fascilin (FAS) 1-1 domain revealing the functional importance of this motif; this was further highlighted by the ability of aa 136-151 peptide to inhibit integrin-mediated cell migration. Immunohistochemistry using MPC5B4, indicated that breast tumour cell POSTN expression was a strong prognostic indicator, along with tumour size, lymph node, and human epidermal growth factor receptor 2 (HER2) status.
Asunto(s)
Anticuerpos Monoclonales , Biomarcadores de Tumor/análisis , Neoplasias de la Mama/patología , Moléculas de Adhesión Celular/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Secuencias de Aminoácidos , Animales , Especificidad de Anticuerpos , Sitios de Unión de Anticuerpos , Neoplasias de la Mama/metabolismo , Movimiento Celular/fisiología , Femenino , Humanos , Inmunohistoquímica , Ratones , Persona de Mediana Edad , Análisis de Matrices TisularesRESUMEN
BACKGROUND: Periostin, an extracellular matrix protein, is expressed in bone, more specifically, the periosteum and periodontal ligaments, and plays a key role in formation and metabolism of bone tissues. Human adipose tissue-derived mesenchymal stem cells (hASCs) have been reported to differentiate into osteoblasts and stimulate bone repair. However, the role of periostin in hASC-mediated bone healing has not been clarified. In the current study, we examined the effect of periostin on bone healing capacity of hASCs in a critical size calvarial defect model. METHODS AND RESULTS: Recombinant periostin protein stimulated migration, adhesion, and proliferation of hASCs in vitro. Implantation of either hASCs or periostin resulted in slight, but not significant, stimulation of bone healing, whereas co-implantation of hASCs together with periostin further potentiated bone healing. In addition, the number of Ki67-positive proliferating cells was significantly increased in calvarial defects by co-implantation of both hASCs and periostin. Consistently, proliferation of administered hASCs was stimulated by co-implantation with periostin in vivo. In addition, co-delivery of hASCs with periostin resulted in markedly increased numbers of CD31-positive endothelial cells and α-SMA-positive arterioles in calvarial defects. CONCLUSIONS: These results suggest that recombinant periostin potentiates hASC-mediated bone healing by stimulating proliferation of transplanted hASCs and angiogenesis in calvarial defects.
Asunto(s)
Regeneración Ósea/efectos de los fármacos , Fosfatos de Calcio/química , Moléculas de Adhesión Celular/farmacología , Durapatita/química , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/efectos de los fármacos , Andamios del Tejido/química , Tejido Adiposo/citología , Adulto , Animales , Adhesión Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Durapatita/farmacología , Femenino , Humanos , Masculino , Trasplante de Células Madre Mesenquimatosas , Ratones , Neovascularización Fisiológica/efectos de los fármacos , Osteoblastos/citología , Osteoblastos/efectos de los fármacos , Cráneo/irrigación sanguínea , Cráneo/citología , Cráneo/efectos de los fármacos , Cráneo/fisiologíaRESUMEN
Prostate cancer is the most frequently diagnosed malignancy and the second leading cause of cancer mortality among men in the United States. Accumulating evidence suggests that lysophosphatidic acid (LPA) serves as an autocrine/paracrine mediator to affect initiation, progression and metastasis of prostate cancer. In the current study, we demonstrate that LPA stimulates migration and proliferation of highly metastatic human prostate cancer, PC-3M-luc-C6 cells. LPA-induced migration of PC-3M-luc-C6 cells was abrogated by pretreatment of PC-3M-luc-C6 cells with the LPA receptor 1/3 inhibitor Ki16425 or small interfering RNA (siRNA)-mediated silencing of endogenous LPA receptor 1, implicating a key role of the LPA-LPA receptor 1 signaling axis in migration of PC-3M-luc-C6 cells. In addition, LPA treatment resulted in augmented expression levels of Krüppel-like factor 4 (KLF4), and siRNA or short-hairpin RNA (shRNA)-mediated silencing of KLF4 expression resulted in the abolishment of LPA-stimulated migration and proliferation of PC-3M-luc-C6 cells. shRNA-mediated silencing of KLF4 expression resulted in the inhibition of in vivo growth of PC-3M-luc-C6 cells in a xenograft transplantation animal model. Taken together, these results suggest a key role of LPA-induced KLF4 expression in cell migration and proliferation of prostate cancer cells in vitro and in vivo.
Asunto(s)
Factores de Transcripción de Tipo Kruppel/metabolismo , Lisofosfolípidos/metabolismo , Próstata/patología , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , Animales , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Silenciador del Gen , Humanos , Factor 4 Similar a Kruppel , Factores de Transcripción de Tipo Kruppel/genética , Masculino , Ratones Endogámicos BALB C , Próstata/metabolismo , Neoplasias de la Próstata/genéticaRESUMEN
Periostin, an extracellular matrix protein, is expressed in injured tissues, such as the heart with myocardial infarction, and promotes angiogenesis and tissue repair. However, the molecular mechanism associated with periostin-stimulated angiogenesis and tissue repair is still unclear. In order to clarify the role of periostin in neovascularization, we examined the effect of periostin in angiogenic potentials of human endothelial colony forming cells (ECFCs) in vitro and in an ischemic limb animal model. Recombinant periostin protein stimulated the migration and tube formation of ECFCs. To identify the functional domains of periostin implicated in angiogenesis, five fragments of periostin, including four repeating FAS-1 domains and a carboxyl terminal domain, were expressed in Escherichia coli and purified to homogeneity. Of the five different domains, the first FAS-1 domain stimulated the migration and tube formation of human ECFCs as potent as the whole periostin. Chemotactic migration of ECFCs induced by the full length and the first FAS-1 domain of periostin was abrogated by blocking antibodies against ß3 and ß5 integrins. Intramuscular injection of the full length and the first FAS-1 domain of periostin into the ischemic hindlimb of mice attenuated severe limb loss and promoted blood perfusion and homing of intravenously administered ECFCs to the ischemic limb. These results suggest that the first FAS-1 domain is responsible for periostin-induced migration and angiogenesis and it can be used as a therapeutic tool for treatment of peripheral artery occlusive disease by stimulating homing of ECFCs.
Asunto(s)
Moléculas de Adhesión Celular Neuronal/metabolismo , Moléculas de Adhesión Celular/metabolismo , Modelos Animales de Enfermedad , Endotelio Vascular/metabolismo , Isquemia/prevención & control , Neovascularización Patológica/prevención & control , Proteínas Recombinantes/metabolismo , Inductores de la Angiogénesis/farmacología , Animales , Western Blotting , Adhesión Celular , Movimiento Celular , Proliferación Celular , Células Cultivadas , Ensayo de Unidades Formadoras de Colonias , Endotelio Vascular/citología , Técnica del Anticuerpo Fluorescente , Miembro Posterior/irrigación sanguínea , Humanos , Inyecciones Intramusculares , Isquemia/metabolismo , Isquemia/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Proteínas Recombinantes/genéticaRESUMEN
Reprogramming of somatic cells to pluripotent cells requires the introduction of factors driving fate switches. Viral delivery has been the most efficient method for generation of induced pluripotent stem cells. Transfection, which precedes virus production, is a commonly-used process for delivery of nucleic acids into cells. The aim of this study is to evaluate the efficiency of PLGA/ bPEI nanoparticles in transfection and virus production. Using a modified method of producing PLGA nanoparticles, PLGA/bPEI-DNA nanoparticles were examined for transfection efficiency and virus production yield in comparison with PLGA-DNA, bPEI-DNA nanoparticles or liposome-DNA complexes. After testing various ratios of PLGA, bPEI, and DNA, the ratio of 6:3:1 (PLGA:bPEI:DNA, w/w/w) was determined to be optimal, with acceptable cellular toxicity. PLGA/bPEI-DNA (6:3:1) nanoparticles showed superior transfection efficiency, especially in multiple gene transfection, and viral yield when compared with liposome-DNA complexes. The culture supernatants of HEK293FT cells transfected with PLGA/bPEI-DNA of viral constructs containing reprogramming factors (Oct4, Sox2, Klf4, or c-Myc) successfully and more efficiently generated induced pluripotent stem cell colonies from mouse embryonic fibroblasts. These results strongly suggest that PLGA/bPEI-DNA nanoparticles can provide significant advantages in studying the effect of multiple factor delivery such as in reprogramming or direct conversion of cell fate.