RESUMEN
Heat shock protein 27 (HSP27) is one of the small molecular chaperones and is involved in many cell mechanisms. Besides the known protective and helpful functions of intracellular HSP27, very little is known about the mode of action of extracellular HSP27. In a previous study, we showed that intravitreal injection of HSP27 led to neuronal damage in the retina and optic nerve after 21 days. However, it was not clear which degenerative signaling pathways were induced by the injection. For this reason, the pathological mechanisms of intravitreal HSP27 injection after 14 days were investigated. Histological and RT-qPCR analyses revealed an increase in endogenous HSP27 in the retina and an activation of components of the intrinsic and extrinsic apoptosis pathway. In addition, an increase in nucleus factor-kappa-light-chain-enhancer of activated B cells (NFκB), as well as of microglia/macrophages and T-cells could be observed. In the optic nerve, however, only an increased apoptosis rate was detectable. Therefore, the activation of caspases and the induction of an incipient immune response seem to be the main triggers for retinal degeneration in this intravitreal HSP27 model.
Asunto(s)
Caspasas/metabolismo , Proteínas de Choque Térmico HSP27/metabolismo , Retina/metabolismo , Linfocitos T/metabolismo , Vías Visuales/metabolismo , Animales , Apoptosis/genética , Caspasas/genética , Regulación de la Expresión Génica , Proteínas de Choque Térmico HSP27/administración & dosificación , Proteínas de Choque Térmico HSP27/genética , Inyecciones Intravítreas , Masculino , Nervio Óptico/metabolismo , Ratas WistarRESUMEN
Heat shock protein 27 (HSP27) is commonly involved in cellular stress. Increased levels of HSP27 as well as autoantibodies against this protein were previously detected in glaucoma patients. Moreover, systemic immunization with HSP27 induced glaucoma-like damage in rodents. Now, for the first time, the direct effects of an intravitreal HSP27 application were investigated. For this reason, HSP27 or phosphate buffered saline (PBS, controls) was applied intravitreally in rats (n = 12/group). The intraocular pressure (IOP) as well as the electroretinogram recordings were comparable in HSP27 and control eyes 21 days after the injection. However, significantly fewer retinal ganglion cells (RGCs) and amacrine cells were observed in the HSP27 group via immunohistochemistry and western blot analysis. The number of bipolar cells, on the other hand, was similar in both groups. Interestingly, a stronger neurofilament degeneration was observed in HSP27 optic nerves, while no differences were noted regarding the myelination state. In summary, intravitreal HSP27 injection led to an IOP-independent glaucoma-like damage. A degeneration of RGCs as well as their axons and amacrine cells was noted. This suggests that high levels of extracellular HSP27 could have a direct damaging effect on RGCs.
Asunto(s)
Proteínas de Choque Térmico HSP27/farmacología , Filamentos Intermedios/efectos de los fármacos , Nervio Óptico/efectos de los fármacos , Retina/efectos de los fármacos , Células Ganglionares de la Retina/efectos de los fármacos , Animales , Proteínas de Unión al Calcio/metabolismo , Electrorretinografía , Proteínas de Choque Térmico HSP27/administración & dosificación , Filamentos Intermedios/metabolismo , Presión Intraocular/efectos de los fármacos , Masculino , Proteínas de la Membrana/metabolismo , Proteínas de Microfilamentos/metabolismo , Microglía/citología , Microglía/efectos de los fármacos , Microglía/metabolismo , Vaina de Mielina/efectos de los fármacos , Vaina de Mielina/metabolismo , Nervio Óptico/metabolismo , Nervio Óptico/fisiología , Ratas Wistar , Retina/metabolismo , Retina/fisiología , Células Ganglionares de la Retina/metabolismoRESUMEN
To improve an effective hepatitis C virus (HCV) therapeutic vaccine, induction of a strong and long term HCV antigen-specific immune response is an important parameter. HCV non-structural protein 3 (NS3) has antigenic properties and plays a major role in viral clearance. In this study, DNA constructs encoding HCV NS3 and heat shock protein 27 (Hsp27)-NS3 genes, and the recombinant (r) NS3 and rHsp27-NS3 proteins complexed with HR9 and Cady-2 cell penetrating peptides (CPPs) were utilized to evaluate antibody, cytokine and Granzyme B secretion in mice. Herein, the formation of NS3 and Hsp27-NS3 DNA/ HR9 CPP complexes were revealed by gel retardation assay and protection against DNase and protease. Cady-2 peptide was used to form the nanoparticles with rNS3 and rHsp27-NS3 proteins. The size and charge of the nanoparticles were confirmed by SEM and Zetasizer instruments. Next, in vitro transfection of the nanoparticles was assessed by flow cytometry and western blotting. Finally, humoral and cellular immune responses were evaluated using different modalities in mice. Our data showed that HR9 and Cady-2 could form stable nanoparticles with DNA and proteins, respectively and enhance their delivery into HEK-293 T cells in a non-covalent approach. Furthermore, the heterologous Hsp27-NS3 DNA + HR9 prime/rHsp27-NS3+Cady-2 protein boost elicited a higher Th1 cellular immune response with a predominant IgG2a, IgG2b, IFN-γ profile and strong Granzyme B secretion than those induced by other groups. Briefly, the combination of a natural adjuvant (Hsp27) and CPPs (HR9 and Cady-2) could significantly stimulate effective immune responses as a promising approach for development of HCV therapeutic vaccines.
Asunto(s)
Hepacivirus/inmunología , Hepatitis C/prevención & control , Vacunas contra Hepatitis Viral/inmunología , Proteínas no Estructurales Virales/inmunología , Adyuvantes Inmunológicos/administración & dosificación , Animales , Péptidos de Penetración Celular/administración & dosificación , Modelos Animales de Enfermedad , Femenino , Células HEK293 , Proteínas de Choque Térmico HSP27/administración & dosificación , Proteínas de Choque Térmico HSP27/genética , Proteínas de Choque Térmico HSP27/inmunología , Hepatitis C/inmunología , Hepatitis C/virología , Humanos , Inmunogenicidad Vacunal , Ratones , Proteínas Recombinantes de Fusión/administración & dosificación , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Vacunas de ADN/administración & dosificación , Vacunas de ADN/inmunología , Vacunas contra Hepatitis Viral/administración & dosificación , Proteínas no Estructurales Virales/administración & dosificación , Proteínas no Estructurales Virales/genéticaRESUMEN
Protein drugs have attracted considerable attention as therapeutic agents due to their diversity and biocompatibility. However, hydrophilic proteins possess difficulty in penetrating lipophilic cell membrane. Although protein transduction domains (PTDs) have shown effectiveness in protein delivery, the importance of selection and position of PTDs in recombinant protein vector constructs has not been investigated. This study intends to investigate the significance of PTD selection and position for therapeutic protein delivery. Heat shock protein 27 (Hsp27) would be a therapeutic protein for the treatment of ischemic heart diseases, but itself is insufficient to prevent systemic degradation and overcoming biochemical barriers during cellular transport. Among all PTD-Hsp27 fusion proteins we cloned, Tat-Hsp27 fusion protein showed the highest efficacy. Nona-arginine (9R) conjugation to the N-terminal of Hsp27 (Hsp27-T) showed higher efficacy than C-terminal. To test the synergistic effect of two PTDs, Tat was inserted to the N-terminal of Hsp27-9R. Tat-Hsp27-9R exhibited enhanced transduction efficiency and significant improvement against oxidative stress and apoptosis. PTD-Hsp27 fusion proteins have strong potential to be developed as therapeutic proteins for the treatment of ischemic heart diseases and selection and position of PTDs for improved efficacy of PTD-fusion proteins need to be optimized considering protein's nature, transduction efficiency and stability.
Asunto(s)
Sistemas de Liberación de Medicamentos/métodos , Proteínas de Choque Térmico HSP27/administración & dosificación , Líquido Intracelular/efectos de los fármacos , Miocitos Cardíacos/efectos de los fármacos , Proteínas Recombinantes de Fusión/administración & dosificación , Animales , Línea Celular , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Relación Dosis-Respuesta a Droga , Composición de Medicamentos/métodos , Proteínas de Choque Térmico HSP27/metabolismo , Humanos , Líquido Intracelular/metabolismo , Miocitos Cardíacos/metabolismo , Dominios Proteicos , Ratas , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
Alzheimer's disease (AD) is an age-related disorder that causes a loss of brain function. Hyperphosphorylation of tau and the subsequent formation of intracellular neurofibrillary tangles (NFTs) are implicated in the pathogenesis of AD. Hyperphosphorylated tau accumulates into insoluble paired helical filaments that aggregate into NFTs; therefore, regulation of tau phosphorylation represents an important treatment approach for AD. Heat shock protein 27 (Hsp27) plays a specific role in human neurodegenerative diseases; however, few studies have examined its therapeutic effect. In this study, we induced tau hyperphosphorylation using okadaic acid, which is a protein phosphatase inhibitor, and generated a fusion protein of Hsp27 and the protein transduction domain of the HIV Tat protein (Tat-Hsp27) to enhance the delivery of Hsp27. We treated Tat-Hsp27 to SH-SY5Y neuroblastoma cells for 2 h; the transduction level was proportional to the Tat-hsp27 concentration. Additionally, Tat-Hsp27 reduced the level of hyperphosphorylated tau and protected cells from apoptotic cell death caused by abnormal tau aggregates. These results reveal that Hsp27 represents a valuable protein therapeutic for AD.
Asunto(s)
Proteínas de Choque Térmico HSP27/administración & dosificación , Neuroblastoma/metabolismo , Ácido Ocadaico/toxicidad , Proteínas Recombinantes de Fusión/administración & dosificación , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/administración & dosificación , Proteínas tau/metabolismo , Línea Celular Tumoral , Relación Dosis-Respuesta a Droga , Proteínas de Choque Térmico , Humanos , Chaperonas Moleculares , Fármacos Neuroprotectores/administración & dosificación , Fosforilación/efectos de los fármacos , Fosforilación/fisiologíaRESUMEN
Heat shock protein 27 (HSP27) exerts cytoprotection against many cellular insults including cerebral ischemia. We previously indicated that intravenous injection of HSP27 purified from human lymphocytes (hHSP27) significantly reduced infarct volume following cerebral ischemia-reperfusion injury, while recombinant HSP27 (rHSP27) was less effective. Phosphorylation is important for HSP27 function, and hHSP27 was more highly phosphorylated than rHSP27. We hypothesized that MAPKAP kinase 2 in vitro-phosphorylated rHSP27 (prHSP27) might increase its brain protection. Mice underwent transient 1-h middle cerebral artery occlusion (MCAO), and then received tail-vein injections of one of the following 1h after reperfusion: hHSP27 as positive control, rHSP27, prHSP27, or bovine serum albumin (BSA) as control. We measured infarct volume, neurological deficits, neurological severity, physiological parameters, cell-death, oxidative stress, and inflammatory response. Compared with BSA controls (30.7±3.1mm(3), n=5), infarct volume was reduced by 67% in the hHSP27 positive-control group (10.1±4.6mm(3), P<0.001, n=5), 17% following rHSP27 (25.4±3.6mm(3), P<0.05, n=5), and 46% following prHSP27 (16.5±4.0mm(3), P<0.001, n=9). Compared to the rHSP27 and BSA-treated groups, prHSP27 also reduced functional deficits, and significantly suppressed apoptosis, oxidative stress, and inflammatory responses. Here, we showed the superior neuroprotective effects of phosphorylated HSP27 by administering prHSP27. prHSP27 may be a useful therapeutic agent to protect against acute cerebral ischemic stroke.
Asunto(s)
Isquemia Encefálica/tratamiento farmacológico , Proteínas de Choque Térmico HSP27/administración & dosificación , Proteínas de Choque Térmico HSP27/metabolismo , Fármacos Neuroprotectores/administración & dosificación , Fármacos Neuroprotectores/metabolismo , Animales , Apoptosis/efectos de los fármacos , Isquemia Encefálica/patología , Muerte Celular/efectos de los fármacos , Encefalitis/metabolismo , Humanos , Infarto de la Arteria Cerebral Media , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Ratones , Ratones Endogámicos C57BL , Fosforilación , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/metabolismoRESUMEN
Heat shock proteins, acting as molecular chaperones, protect heart muscle from ischemic injury and offer a potential approach to therapy. Here we describe preparation of an injectable form of heat shock protein 27, fused with a protein transduction domain (TAT-HSP27) and contained in a hybrid system of poly(d,l-lactic-co-glycolic acid) microsphere and alginate hydrogel. By varying the porous structure of the microspheres, the release of TAT-HSP27 from the hybrid system was sustained for two weeks in vitro. The hybrid system containing TAT-HSP27 was intramyocardially injected into a murine myocardial infarction model, and its therapeutic effect was evaluated in vivo. The sustained delivery of TAT-HSP27 substantially suppressed apoptosis in the infarcted site, and improved the ejection fraction, end-systolic volume and maximum pressure development in the heart. Local and sustained delivery of anti-apoptotic proteins such as HSP27 using a hybrid system may present a promising approach to the treatment of ischemic diseases.
Asunto(s)
Alginatos/química , Portadores de Fármacos/química , Productos del Gen tat/uso terapéutico , Proteínas de Choque Térmico HSP27/uso terapéutico , Ácido Láctico/química , Infarto del Miocardio/tratamiento farmacológico , Ácido Poliglicólico/química , Proteínas Recombinantes de Fusión/uso terapéutico , Animales , Apoptosis/efectos de los fármacos , Preparaciones de Acción Retardada , Modelos Animales de Enfermedad , Productos del Gen tat/administración & dosificación , Productos del Gen tat/farmacocinética , Ácido Glucurónico/química , Proteínas de Choque Térmico HSP27/administración & dosificación , Proteínas de Choque Térmico HSP27/farmacocinética , Ácidos Hexurónicos/química , Hidrogeles , Etiquetado Corte-Fin in Situ , Masculino , Microesferas , Infarto del Miocardio/metabolismo , Infarto del Miocardio/patología , Copolímero de Ácido Poliláctico-Ácido Poliglicólico , Ratas , Ratas Sprague-Dawley , Proteínas Recombinantes de Fusión/administración & dosificación , Proteínas Recombinantes de Fusión/farmacocinéticaRESUMEN
Although challenging, neuroprotective therapies for ischemic stroke remain an interesting strategy for countering ischemic injury and suppressing brain tissue damage. Among potential neuroprotective molecules, heat shock protein 27 (HSP27) is a strong cell death suppressor. To assess the neuroprotective effects of HSP27 in a mouse model of transient middle cerebral artery occlusion, we purified a "physiological" HSP27 (hHSP27) from normal human lymphocytes. hHSP27 differed from recombinant HSP27 in that it formed dimeric, tetrameric, and multimeric complexes, was phosphorylated, and contained small amounts of αß-crystallin and HSP20. Mice received intravenous injections of hHSP27 following focal cerebral ischemia. Infarct volume, neurological deficit scores, physiological parameters, and immunohistochemical analyses were evaluated 24 h after reperfusion. Intravenous injections of hHSP27 1 h after reperfusion significantly reduced infarct size and improved neurological deficits. Injected hHSP27 was localized in neurons on the ischemic side of the brain. hHSP27 suppressed neuronal cell death resulting from cytochrome c-mediated caspase activation, oxidative stress, and inflammatory responses. Recombinant HSP27 (rHSP27), which was artificially expressed and purified from Escherichia coli, and dephosphorylated hHSP27 did not have brain protective effects, suggesting that the phosphorylation of hHSP27 may be important for neuroprotection after ischemic insults. The present study suggests that hHSP27 with posttranslational modifications provided neuroprotection against ischemia/reperfusion injury and that the protection was mediated through the inhibition of apoptosis, oxidative stress, and inflammation. Intravenously injected human HSP27 should be explored for the treatment of acute ischemic strokes.
Asunto(s)
Isquemia Encefálica/metabolismo , Proteínas de Choque Térmico HSP27/metabolismo , Fármacos Neuroprotectores/metabolismo , Accidente Cerebrovascular/metabolismo , Accidente Cerebrovascular/prevención & control , Animales , Apoptosis/efectos de los fármacos , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Encéfalo/patología , Infarto Encefálico/metabolismo , Infarto Encefálico/prevención & control , Isquemia Encefálica/patología , Daño del ADN/efectos de los fármacos , Modelos Animales de Enfermedad , Proteínas de Choque Térmico HSP27/administración & dosificación , Proteínas de Choque Térmico HSP27/aislamiento & purificación , Humanos , Peroxidación de Lípido/efectos de los fármacos , Masculino , Ratones , Neuroglía/efectos de los fármacos , Neuroglía/metabolismo , Fármacos Neuroprotectores/administración & dosificación , Fármacos Neuroprotectores/aislamiento & purificación , Estrés Oxidativo/efectos de los fármacos , Fosforilación , Daño por Reperfusión/metabolismo , Daño por Reperfusión/prevención & controlRESUMEN
Ischemic disease is associated with high mortality and morbidity rates, and therapeutic angiogenesis via systemic or local delivery of protein drugs is one potential approach to treat the disease. In this study, we hypothesized that combined delivery of TAT-HSP27 (HSP27 fused with transcriptional activator) and VEGF could enhance the therapeutic efficacy in an ischemic mouse model, and that sequential release could be critical in therapeutic angiogenesis. Alginate hydrogels containing TAT-HSP27 as an anti-apoptotic agent were prepared, and porous PLGA microspheres loaded with VEGF as an angiogenic agent were incorporated into the hydrogels to prepare microsphere/hydrogel hybrid delivery systems. Sequential in vitro release of TAT-HSP27 and VEGF was achieved by the hybrid systems. TAT-HSP27 was depleted from alginate gels in 7 days, while VEGF was continually released for 28 days. The release rate of VEGF was attenuated by varying the porous structures of PLGA microspheres. Sequential delivery of TAT-HSP27 and VEGF was critical to protect against muscle degeneration and fibrosis, as well as to promote new blood vessel formation in the ischemic site of a mouse model. This approach to controlling the sequential release behaviors of multiple drugs could be useful in the design of novel drug delivery systems for therapeutic angiogenesis.
Asunto(s)
Portadores de Fármacos/química , Productos del Gen tat/administración & dosificación , Proteínas de Choque Térmico HSP27/administración & dosificación , Hidrogeles/química , Isquemia/tratamiento farmacológico , Neovascularización Fisiológica/efectos de los fármacos , Proteínas Recombinantes de Fusión/administración & dosificación , Factor A de Crecimiento Endotelial Vascular/administración & dosificación , Alginatos/química , Animales , Apoptosis/efectos de los fármacos , Modelos Animales de Enfermedad , Combinación de Medicamentos , Arteria Femoral/efectos de los fármacos , Arteria Femoral/patología , Productos del Gen tat/farmacología , Productos del Gen tat/uso terapéutico , Proteínas de Choque Térmico HSP27/farmacología , Proteínas de Choque Térmico HSP27/uso terapéutico , Miembro Posterior/irrigación sanguínea , Miembro Posterior/efectos de los fármacos , Miembro Posterior/patología , Etiquetado Corte-Fin in Situ , Isquemia/metabolismo , Ácido Láctico/química , Ratones , Ratones Desnudos , Microscopía Electrónica de Rastreo , Microesferas , Tamaño de la Partícula , Ácido Poliglicólico/química , Copolímero de Ácido Poliláctico-Ácido Poliglicólico , Porosidad , Proteínas Recombinantes de Fusión/farmacología , Proteínas Recombinantes de Fusión/uso terapéutico , Solubilidad , Propiedades de Superficie , Factor A de Crecimiento Endotelial Vascular/farmacología , Factor A de Crecimiento Endotelial Vascular/uso terapéuticoRESUMEN
Myocardial infarction causes a high rate of morbidity and mortality worldwide, and heat shock proteins as molecular chaperones have been attractive targets for protecting cardiomyoblasts under environmental stimuli. In this study, in order to enhance the penetration of heat shock protein 27 (HSP27) across cell membranes, we fused HSP27 with transcriptional activator (TAT) derived from human immunodeficiency virus (HIV) as a protein transduction domain (PTD). We loaded the fusion protein (TAT-HSP27) into microsphere/hydrogel combination delivery systems to control the release behavior for prolonged time periods. We found that the release behavior of TAT-HSP27 was able to be controlled by varying the ratio of PLGA microspheres and alginate hydrogels. Indeed, the released fusion protein maintained its bioactivity and could recover the proliferation of cardiomyoblasts cultured under hypoxic conditions. This approach to controlling the release behavior of TAT-HSP27 using microsphere/hydrogel combination delivery systems may be useful for treating myocardial infarction in a minimally invasive manner.