RESUMEN
Opticin is an endogenous vitreous glycoprotein that may have therapeutic potential as it has been shown that supranormal concentrations suppress preretinal neovascularization. Herein we investigated the pharmacokinetics of opticin following intravitreal injection in rabbits. To measure simultaneously concentrations of human and rabbit opticin, a selected reaction monitoring mass spectrometry assay was developed. The mean concentration of endogenous rabbit opticin in 7 uninjected eyes was measured and found to be 19.2 nM or 0.62 µg/mL. When the vitreous was separated by centrifugation into a supernatant and collagen-containing pellet, 94% of the rabbit opticin was in the supernatant. Intravitreal injection of human opticin (40 µg) into both eyes of rabbits was followed by enucleation at 5, 24, and 72 h and 7, 14, and 28 days postinjection (n = 6 at each time point) and measurement of vitreous human and rabbit opticin concentrations in the supernatant and collagen-containing pellet following centrifugation. The volume of distribution of human opticin was calculated to be 3.31 mL, and the vitreous half-life was 4.2 days. Assuming that rabbit and human opticin are cleared from rabbit vitreous at the same rate, opticin is secreted into the vitreous at a rate of 0.14 µg/day. We conclude that intravitreally injected opticin has a vitreous half-life that is similar to currently available antiangiogenic therapeutics. While opticin was first identified bound to vitreous collagen fibrils, here we demonstrate that >90% of endogenous opticin is not bound to collagen. Endogenous opticin is secreted by the nonpigmented ciliary epithelium into the rabbit vitreous at a remarkably high rate, and the turnover in vitreous is approximately 15% per day.
Asunto(s)
Inhibidores de la Angiogénesis/administración & dosificación , Inhibidores de la Angiogénesis/farmacocinética , Proteínas de la Matriz Extracelular/administración & dosificación , Proteínas de la Matriz Extracelular/farmacocinética , Inyecciones Intravítreas/métodos , Proteoglicanos/administración & dosificación , Proteoglicanos/farmacocinética , Inhibidores de la Angiogénesis/biosíntesis , Animales , Colágeno/metabolismo , Proteínas de la Matriz Extracelular/biosíntesis , Proteínas de la Matriz Extracelular/metabolismo , Semivida , Humanos , Masculino , Espectrometría de Masas/métodos , Neovascularización Fisiológica/efectos de los fármacos , Proteoglicanos/biosíntesis , Proteoglicanos/metabolismo , Conejos , Retina/metabolismo , Cuerpo Vítreo/metabolismoRESUMEN
Triple-negative breast cancer (TNBC) patients have the worst prognosis and distant metastasis-free survival among all major subtypes of breast cancer. The poor clinical outlook is further exacerbated by a lack of effective targeted therapies for TNBC. Here we show that ectopic expression and therapeutic delivery of the secreted protein Tubulointerstitial nephritis antigen-like 1 (Tinagl1) suppresses TNBC progression and metastasis through direct binding to integrin α5ß1, αvß1, and epidermal growth factor receptor (EGFR), and subsequent simultaneous inhibition of focal adhesion kinase (FAK) and EGFR signaling pathways. Moreover, Tinagl1 protein level is associated with good prognosis and reversely correlates with FAK and EGFR activation status in TNBC. Our results suggest Tinagl1 as a candidate therapeutic agent for TNBC by dual inhibition of integrin/FAK and EGFR signaling pathways.
Asunto(s)
Proteínas de la Matriz Extracelular/genética , Integrina alfa5beta1/metabolismo , Lipocalinas/genética , Neoplasias Pulmonares/terapia , Receptores de Vitronectina/metabolismo , Neoplasias de la Mama Triple Negativas/terapia , Animales , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Progresión de la Enfermedad , Receptores ErbB/metabolismo , Proteínas de la Matriz Extracelular/administración & dosificación , Proteínas de la Matriz Extracelular/metabolismo , Femenino , Quinasa 1 de Adhesión Focal/metabolismo , Regulación Neoplásica de la Expresión Génica , Humanos , Lipocalinas/administración & dosificación , Lipocalinas/metabolismo , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/secundario , Ratones , Pronóstico , Transducción de Señal , Neoplasias de la Mama Triple Negativas/genética , Neoplasias de la Mama Triple Negativas/metabolismoRESUMEN
BACKGROUND: Spermatogonial stem cell transplantation (SSCT) could become a fertility restoration tool for childhood cancer survivors. However, since in mice, the colonization efficiency of transplanted spermatogonial stem cells (SSCs) is only 12%, the efficiency of the procedure needs to be improved before clinical implementation is possible. Co-transplantation of mesenchymal stem cells (MSCs) might increase colonization efficiency of SSCs by restoring the SSC niche after gonadotoxic treatment. METHODS: A mouse model for long-term infertility was developed and used to transplant SSCs (SSCT, n = 10), MSCs (MSCT, n = 10), a combination of SSCs and MSCs (MS-SSCT, n = 10), or a combination of SSCs and TGFß1-treated MSCs (MSi-SSCT, n = 10). RESULTS: The best model for transplantation was obtained after intraperitoneal injection of busulfan (40 mg/kg body weight) at 4 weeks followed by CdCl2 (2 mg/kg body weight) at 8 weeks of age and transplantation at 11 weeks of age. Three months after transplantation, spermatogenesis resumed with a significantly better tubular fertility index (TFI) in all transplanted groups compared to non-transplanted controls (P < 0.001). TFI after MSi-SSCT (83.3 ± 19.5%) was significantly higher compared to MS-SSCT (71.5 ± 21.7%, P = 0.036) but did not differ statistically compared to SSCT (78.2 ± 12.5%). In contrast, TFI after MSCT (50.2 ± 22.5%) was significantly lower compared to SSCT (P < 0.001). Interestingly, donor-derived TFI was found to be significantly improved after MSi-SSCT (18.8 ± 8.0%) compared to SSCT (1.9 ± 1.1%; P < 0.001), MSCT (0.0 ± 0.0%; P < 0.001), and MS-SSCT (3.4 ± 1.9%; P < 0.001). While analyses showed that both native and TGFß1-treated MSCs maintained characteristics of MSCs, the latter showed less migratory characteristics and was not detected in other organs. CONCLUSION: Co-transplanting SSCs and TGFß1-treated MSCs significantly improves the recovery of endogenous SSCs and increases the homing efficiency of transplanted SSCs. This procedure could become an efficient method to treat infertility in a clinical setup, once the safety of the technique has been proven.
Asunto(s)
Células Madre Germinales Adultas/trasplante , Infertilidad Masculina/terapia , Trasplante de Células Madre Mesenquimatosas , Animales , Busulfano/administración & dosificación , Cloruro de Cadmio/administración & dosificación , Supervivencia Celular , Modelos Animales de Enfermedad , Proteínas de la Matriz Extracelular/administración & dosificación , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Espermatogénesis , Factor de Crecimiento Transformador beta/administración & dosificaciónRESUMEN
BACKGROUND: The degeneration of the intervertebral disc (IVD) is characterized by proteolytic degradation of the extracellular matrix, and its repair requires the production of an extracellular matrix with a high proteoglycan-to-collagen ratio characteristic of a nucleus pulposus (NP)-like phenotype in vivo. At the moment, there is no medical treatment to reverse or even retard disc degeneration. The purpose of the present study was to determine if a low dose of short link N (sLN), a recently discovered fragment of the link N peptide, could behave in a manner similar to that of link N in restoring the proteoglycan content and proteoglycan-to-collagen ratio of the disc in a rabbit model of IVD degeneration, as an indication of its potential therapeutic benefit in reversing disc degeneration. METHODS: Adolescent New Zealand white rabbits received an annular puncture with an 18-gauge needle into two noncontiguous discs to induce disc degeneration. Two weeks later, either saline (10 µL) or sLN (25 µg in 10 µL saline) was injected into the center of the NP. The sLN concentration was empirically chosen at a lower molar concentration equivalent to half that of link N (100 µg in 10 µL). The effect on radiographic, biochemical and histologic changes were evaluated. RESULTS: Following needle puncture, disc height decreased by about 25-30% within 2 weeks and maintained this loss for the duration of the 12-week study; a single 25-µg sLN injection at 2 weeks partially restored this loss in disc height. sLN injection led to an increase in glycosaminoglycans (GAG) content 12 weeks post-injection in both the NP and annulus fibrosus (AF). There was a trend towards maintaining control disc collagen-content with sLN supplementation and the GAG-to-collagen ratio in the NP was increased when compared to the saline group. CONCLUSIONS: When administered to the degenerative disc in vivo, sLN injection leads to an increase in proteoglycan content and a trend towards maintaining control disc collagen content in both the NP and AF. This is similar to link N when it is administered to the degenerative disc. Thus, pharmacologically, sLN supplementation could be a novel therapeutic approach for treating disc degeneration.
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Modelos Animales de Enfermedad , Proteínas de la Matriz Extracelular/farmacología , Degeneración del Disco Intervertebral/tratamiento farmacológico , Disco Intervertebral/efectos de los fármacos , Péptidos/farmacología , Proteoglicanos/farmacología , Secuencia de Aminoácidos , Animales , Colágeno/metabolismo , Proteínas de la Matriz Extracelular/administración & dosificación , Proteínas de la Matriz Extracelular/química , Glicosaminoglicanos/metabolismo , Humanos , Inyecciones , Disco Intervertebral/metabolismo , Disco Intervertebral/patología , Péptidos/administración & dosificación , Proteoglicanos/administración & dosificación , Proteoglicanos/química , Proteoglicanos/metabolismo , Conejos , Resultado del TratamientoRESUMEN
Hypertrophic scarring is a major postoperative complication which leads to severe disfigurement and dysfunction in patients and usually requires multiple surgical revisions due to its high recurrence rates. Excessive-mechanical-loading across wounds is an important initiator of hypertrophic scarring formation. In this study, we demonstrate that intradermal administration of a single extracellular matrix (ECM) molecule-fibromodulin (FMOD) protein-can significantly reduce scar size, increase tensile strength, and improve dermal collagen architecture organization in the normal and even excessive-mechanical-loading red Duroc pig wound models. Since pig skin is recognized by the Food and Drug Administration as the closest animal equivalent to human skin, and because red Duroc pigs show scarring that closely resembles human proliferative scarring and hypertrophic scarring, FMOD-based technologies hold high translational potential and applicability to human patients suffering from scarring-especially hypertrophic scarring.
Asunto(s)
Cicatriz/tratamiento farmacológico , Fibromodulina/administración & dosificación , Enfermedades de la Piel/tratamiento farmacológico , Cicatrización de Heridas/efectos de los fármacos , Animales , Cicatriz/genética , Cicatriz/patología , Proteínas de la Matriz Extracelular/administración & dosificación , Proteínas de la Matriz Extracelular/genética , Fibromodulina/genética , Humanos , Inyecciones Intradérmicas , Piel/efectos de los fármacos , Piel/lesiones , Enfermedades de la Piel/genética , Enfermedades de la Piel/patología , Estrés Mecánico , Porcinos , Resistencia a la Tracción/efectos de los fármacos , Cicatrización de Heridas/genéticaRESUMEN
Duchenne muscular dystrophy (DMD) is a devastating muscle disease caused by loss-of-function mutations in DMD encoding dystrophin. No rational therapy is currently available. Utrophin is a paralog of dystrophin and is highly expressed at the neuromuscular junction. In mdx mice, utrophin is naturally upregulated throughout the muscle fibers, which mitigates muscular dystrophy. Protein-anchoring therapy was previously reported, in which a recombinant extracellular matrix (ECM) protein is delivered to and anchored to a specific target using its proprietary binding domains. Being prompted by a report that intramuscular and intraperitoneal injection of an ECM protein, biglycan, upregulates expression of utrophin and ameliorates muscle pathology in mdx mice, protein-anchoring therapy was applied to mdx mice. Recombinant adeno-associated virus serotype 8 (rAAV8) carrying hBGN encoding human biglycan was intravenously injected into 5-week-old mdx mice. The rAAV8-hBGN treatment improved motor deficits and decreased plasma creatine kinase activities. In muscle sections of treated mice, the number of central myonuclei and the distribution of myofiber sizes were improved. The treated mice increased gene expressions of utrophin and ß1-syntrophin, as well as protein expressions of biglycan, utrophin, γ-sarcoglycan, dystrobrevin, and α1-syntrophin. The expression of hBGN in the skeletal muscle of the treated mice was 1.34-fold higher than that of the native mouse Bgn (mBgn). The low transduction efficiency and improved motor functions suggest that biglycan expressed in a small number of muscle fibers was likely to have been secreted and anchored to the cell surface throughout the whole muscular fibers. It is proposed that the protein-anchoring strategy can be applied not only to deficiency of an ECM protein as previously reported, but also to augmentation of a naturally induced ECM protein.
Asunto(s)
Biglicano/administración & dosificación , Proteínas de la Matriz Extracelular/administración & dosificación , Distrofia Muscular Animal/terapia , Distrofia Muscular de Duchenne/terapia , Animales , Biglicano/genética , Modelos Animales de Enfermedad , Proteínas Asociadas a la Distrofina/genética , Proteínas de la Matriz Extracelular/genética , Regulación de la Expresión Génica , Humanos , Ratones , Ratones Endogámicos mdx , Músculo Esquelético/metabolismo , Músculo Esquelético/patología , Distrofia Muscular Animal/genética , Distrofia Muscular Animal/patología , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/patología , Utrofina/genéticaRESUMEN
After peripheral nerve injury, motor and sensory axons are able to regenerate but inaccuracy of target reinnervation leads to poor functional recovery. Extracellular matrix (ECM) components and neurotrophic factors (NTFs) exert their effect on different neuronal populations creating a suitable environment to promote axonal growth. Here, we assessed in vitro and in vivo the selective effects of combining different ECM components with NTFs on motor and sensory axons regeneration and target reinnervation. Organotypic cultures with collagen, laminin and nerve growth factor (NGF)/neurotrophin-3 (NT3) or collagen, fibronectin and brain-derived neurotrophic factor (BDNF) selectively enhanced sensory neurite outgrowth of DRG neurons and motor neurite outgrowth from spinal cord slices respectively. For in vivo studies, the rat sciatic nerve was transected and repaired with a silicone tube filled with a collagen and laminin matrix with NGF/NT3 encapsulated in poly(lactic-co-glycolic acid) (PLGA) microspheres (MP) (LM + MP.NGF/NT3), or a collagen and fibronectin matrix with BDNF in PLGA MPs (FN + MP.BDNF). Retrograde labeling and functional tests showed that LM + MP.NGF/NT3 increased the number of regenerated sensory neurons and improved sensory functional recovery, whereas FN + MP.BDNF preferentially increased regenerated motoneurons and enhanced motor functional recovery. Therefore, combination of ECM molecules with NTFs may be a good approach to selectively enhance motor and sensory axons regeneration and promote appropriate target reinnervation.
Asunto(s)
Axones/fisiología , Proteínas de la Matriz Extracelular/farmacología , Neuronas Motoras/fisiología , Factores de Crecimiento Nervioso/farmacología , Regeneración Nerviosa/efectos de los fármacos , Traumatismos de los Nervios Periféricos/tratamiento farmacológico , Células Receptoras Sensoriales/fisiología , Animales , Células Cultivadas , Proteínas de la Matriz Extracelular/administración & dosificación , Proteínas de la Matriz Extracelular/uso terapéutico , Femenino , Microesferas , Factores de Crecimiento Nervioso/administración & dosificación , Factores de Crecimiento Nervioso/uso terapéutico , Ratas , Ratas Sprague-DawleyRESUMEN
In this work the effect of RHAMM (receptor hyaluronan-mediated motility)-target peptides was investigated on the viability, apoptosis and necrosis of prostate cancer cells (PC3m-LN4). It has been established that RHAMM-target peptides inhibited on 90 % cell viability of PC3m-LN4 cells at a concentration of 10 ug / ml (2Ñ 10-7 M) for 48 h. It has shown that RHAMM-target peptides induced apoptosis and inhibited necrosis of tumor cells. RHAMM-target peptide had no effect on fibroblasts (non-tumor cells) and fibroblasts (RHAMM-/-). The studies also revealed that RHAMM-target peptides enhanced activity of caspase-3/7 in cancer cells.
Asunto(s)
Proliferación Celular/efectos de los fármacos , Proteínas de la Matriz Extracelular/genética , Receptores de Hialuranos/genética , Péptidos/administración & dosificación , Neoplasias de la Próstata/genética , Apoptosis/efectos de los fármacos , Caspasa 3/genética , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Proteínas de la Matriz Extracelular/administración & dosificación , Fibroblastos/efectos de los fármacos , Humanos , Receptores de Hialuranos/administración & dosificación , Masculino , Péptidos/genética , Neoplasias de la Próstata/patología , Neoplasias de la Próstata/terapia , TransfecciónRESUMEN
Nanofiber-based hydrogels (nanogels) with different, covalently bound peptides were used as an extracellular environment for lens epithelial cells (LECs) in order to modulate the capsular opacification (CO) response after lens surgery in a porcine eye model. Lenses were divided into 15 groups (n = 4 per group), the lens content was removed and the empty capsules were refilled with nanogel without peptides and nanogels with 13 combinations of 5 different peptides: two laminin-derived, two fibronectin-derived, and one collagen IV-derived peptide representing cell adhesion motifs. A control group of 4 lenses was refilled with hyaluronan. After refilling, lenses were extracted from the porcine eye and cultured for three weeks. LECs were assessed for morphology and alpha smooth muscle actin (αSMA) expression using confocal laser scanning microscopy. Compared to hyaluronan controls, lenses filled with nanogel had less CO formation, indicated by a lower αSMA expression (P = 0.004). Microscopy showed differences in morphological cell response within the nanogel refilled groups. αSMA expression in these groups was highest in lenses refilled with nanogel without peptides (9.54 ± 11.29%). Overall, LEC transformation is reduced by the presence of nanogels and the response is improved even further by incorporation of extracellular matrix peptides representing adhesion motifs. Thus, nanomaterials targeting biological pathways, in our case interactions with integrin signaling, are a promising avenue toward reduction of CO. Further research is needed to optimize nanogel-peptide combinations that fully prevent CO.
Asunto(s)
Opacificación Capsular/prevención & control , Células Epiteliales/citología , Transición Epitelial-Mesenquimal/efectos de los fármacos , Proteínas de la Matriz Extracelular/administración & dosificación , Hidrogeles , Cápsula del Cristalino/citología , Oligopéptidos/administración & dosificación , Actinas/metabolismo , Animales , Biomarcadores/metabolismo , Opacificación Capsular/patología , Colágeno Tipo IV/administración & dosificación , Colágeno Tipo IV/síntesis química , Sistemas de Liberación de Medicamentos , Proteínas de la Matriz Extracelular/síntesis química , Fibronectinas/administración & dosificación , Fibronectinas/síntesis química , Técnica del Anticuerpo Fluorescente Indirecta , Laminina/administración & dosificación , Laminina/síntesis química , Cristalino/citología , Nanofibras , Oligopéptidos/síntesis química , Técnicas de Cultivo de Órganos , Sus scrofaRESUMEN
The Reelin signaling pathway is implicated in processes controlling synaptic plasticity and hippocampus-dependent learning and memory. A single direct in vivo application of Reelin enhances long-term potentiation, increases dendritic spine density and improves associative and spatial learning and memory. Angelman syndrome (AS) is a neurological disorder that presents with an overall defect in synaptic function, including decreased long-term potentiation, reduced dendritic spine density, and deficits in learning and memory, making it an attractive model in which to examine the ability of Reelin to recover synaptic function and cognitive deficits. In this study, we investigated the effects of Reelin administration on synaptic plasticity and cognitive function in a mouse model of AS and demonstrated that bilateral, intraventricular injections of Reelin recover synaptic function and corresponding hippocampus-dependent associative and spatial learning and memory. Additionally, we describe alteration of the Reelin profile in tissue from both the AS mouse and post-mortem human brain.
Asunto(s)
Síndrome de Angelman/fisiopatología , Síndrome de Angelman/psicología , Moléculas de Adhesión Celular Neuronal/administración & dosificación , Proteínas de la Matriz Extracelular/administración & dosificación , Hipocampo/efectos de los fármacos , Potenciación a Largo Plazo/efectos de los fármacos , Proteínas del Tejido Nervioso/administración & dosificación , Serina Endopeptidasas/administración & dosificación , Síndrome de Angelman/tratamiento farmacológico , Animales , Moléculas de Adhesión Celular Neuronal/metabolismo , Corteza Cerebral/metabolismo , Espinas Dendríticas/efectos de los fármacos , Modelos Animales de Enfermedad , Proteínas de la Matriz Extracelular/metabolismo , Femenino , Células HEK293 , Hipocampo/fisiopatología , Hipocampo/ultraestructura , Humanos , Inyecciones Intraventriculares , Masculino , Ratones , Actividad Motora/efectos de los fármacos , Proteínas del Tejido Nervioso/metabolismo , Proteína Reelina , Serina Endopeptidasas/metabolismo , Aprendizaje Espacial/efectos de los fármacos , Memoria Espacial/efectos de los fármacosRESUMEN
Reelin has recently attracted attention because of its connection to several neuropsychiatric diseases. We previously reported the finding that prior transplantation of GABAergic neuron precursor cells into the medial prefrontal cortex (mPFC) of mice significantly prevented the induction of cognitive and sensory-motor gating deficits induced by phencyclidine (PCP). The majority of the precursor cells transplanted into the mPFC of the recipient mice differentiated into members of a somatostatin/Reelin-expressing class of GABAergic interneurons. These findings raised the possibility that Reelin secreted by the transplanted cells plays an important role in preventing the deficits induced by PCP. In this study, we investigated whether Reelin itself has a preventive effect on PCP-induced behavioral phenotypes by injecting conditioned medium containing Reelin into the lateral ventricle of the brains of 6- to 7-week-old male mice before administrating PCP. Behavioral analyses showed that the prior Reelin injection had a preventive effect against induction of the cognitive and sensory-motor gating deficits associated with PCP. Moreover, one of the types of Reelin receptor was found to be expressed by neurons in the mPFC. The results of this study point to the Reelin signaling pathway as a candidate target for the pharmacologic treatment of neuropsychiatric diseases.
Asunto(s)
Moléculas de Adhesión Celular Neuronal/administración & dosificación , Moléculas de Adhesión Celular Neuronal/metabolismo , Trastornos del Conocimiento/prevención & control , Proteínas de la Matriz Extracelular/administración & dosificación , Proteínas de la Matriz Extracelular/metabolismo , Proteínas del Tejido Nervioso/administración & dosificación , Proteínas del Tejido Nervioso/metabolismo , Filtrado Sensorial/efectos de los fármacos , Serina Endopeptidasas/administración & dosificación , Serina Endopeptidasas/metabolismo , Animales , Trastornos del Conocimiento/inducido químicamente , Neuronas GABAérgicas , Infusiones Intraventriculares , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos ICR , Neuronas/metabolismo , Fenciclidina/toxicidad , Corteza Prefrontal/metabolismo , Receptores de LDL/metabolismo , Proteína ReelinaRESUMEN
cyclicCHAD is a peptide representing the α2ß1 integrin binding sequence of the matrix protein chondroadherin (CHAD), which in our hands proved effective at counteracting bone loss in ovariectomised mice by inhibiting osteoclastogenesis. Given that bone metastases are characterised by exacerbated osteoclast activity as well, we tested this therapy in mice intracardiacally injected with the osteotropic human breast cancer cell line MDA-MB-231. Treatment with cyclicCHAD significantly decreased cachexia and incidence of bone metastases, and induced a trend of reduction of visceral metastasis volume, while in orthotopically injected mice cyclicCHAD reduced tumour volume. In vitro studies showed its ability to impair tumour cell motility and invasion, suggesting a direct effect not only on osteoclasts but also on the tumour cell phenotype. Interestingly, when administered together with a suboptimal, poorly effective, dose of doxorubicin (DXR), cyclicCHAD improved survival and reduced visceral metastases volume to a level similar to that of the optimal dose of DXR alone. Taken together, these preclinical data suggest that cyclicCHAD is a new inhibitor of bone metastases, with an appreciable direct effect also on tumour growth and a synergistic activity in combination with low dose chemotherapy, underscoring an important translational impact.
Asunto(s)
Neoplasias Óseas/tratamiento farmacológico , Neoplasias de la Mama/tratamiento farmacológico , Proteínas de la Matriz Extracelular/metabolismo , Integrina alfa2beta1/metabolismo , Animales , Neoplasias Óseas/patología , Neoplasias Óseas/secundario , Resorción Ósea/tratamiento farmacológico , Resorción Ósea/patología , Neoplasias de la Mama/patología , Caquexia/tratamiento farmacológico , Línea Celular Tumoral , Doxorrubicina/administración & dosificación , Proteínas de la Matriz Extracelular/administración & dosificación , Femenino , Humanos , Ratones , Osteoclastos/efectos de los fármacos , Osteoclastos/patología , Estructura Terciaria de ProteínaRESUMEN
Ischemic stroke is one of the leading causes of morbidity and mortality worldwide and characterized by defective angiogenesis. The functional sequences (RGDs, GRGDSPASSPISC) derived from fibronectin have been confirmed to augment angiogenesis in vivo and in vitro. However, delivery of peptides into the brain parenchyma has been hampered by the presence of the blood-brain barrier (BBB). We fused RGDs with penetratin (Antp) derived from Drosophila antennapedia homeodomain protein to improve the penetration of peptides through BBB into ischemic hemisphere. We found Antp-RGDs successfully not only penetrate the SH-SY5Y cells but also penetrated through BBB into ischemic hemisphere by intraperitoneal injection. In addition, application of Antp-RGDs to the focal cerebral ischemic reperfusion injury in rats resulted in the reduction of cerebral ischemic volume and the improvement of neurologic score according to the 21-point score. We further demonstrated that activation of phosphorylation-extracellular-signal related kinase 1/2 (p-ERK 1/2) and upregulation of gene VEGF resulted from post-treatment with Antp-RGDs 2 hours after reperfusion onset might at least partly contribute to the benefic changes after focal cerebral ischemic reperfusion injury in rats. Our data suggested that Antp-RGDs may serve as an attractive therapeutic intervention for treating ischemic stroke.
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Proteínas de la Matriz Extracelular/uso terapéutico , Enfermedades del Sistema Nervioso/etiología , Enfermedades del Sistema Nervioso/prevención & control , Oligopéptidos/uso terapéutico , Daño por Reperfusión/complicaciones , Daño por Reperfusión/tratamiento farmacológico , Animales , Barrera Hematoencefálica/metabolismo , Línea Celular , Proteínas de la Matriz Extracelular/administración & dosificación , Fibronectinas/uso terapéutico , Etiquetado Corte-Fin in Situ , Ataque Isquémico Transitorio/metabolismo , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Masculino , Arteria Cerebral Media/fisiopatología , Ratas , Ratas Sprague-Dawley , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
BACKGROUND: Vaccines are the most effective agents to control infections. However, recombinant vaccines often do not elicit strong immune responses. Protein antigens combined with proper adjuvants have been widely used to induce immune responses, especially the humoral immune responses, against various pathogens, including parasites. The extracellular matrix protein mindin has been recognised as an immune facilitator for initiating innate immune responses. It has therefore been expected to be a potentially potent adjuvant in the development of novel vaccines. The aim of this study was to investigate whether mindin could facilitate the induction of antigen-specific immune responses to recombinant antigens (rBAG1, rSRS4 and rSRS9) of Toxoplasma gondii in BALB/c mice. METHODS: In this study, we explored the adjuvant effect of the recombinant mindin in the generation of specific Th1 and Th2 responses to each of three T. gondii antigens, BAG1, SRS4 and SRS9. All mice in the experimental groups received either antigen alone or in combination with Freund's adjuvant or with the recombinant mindin. The immune responses after immunisation were measured by ELISA and lymphoproliferative assays. The immunised mice were challenged with live T. gondii tachyzoites, and the protection efficiency was compared between the groups. RESULTS: Our results revealed that mindin as an adjuvant could facilitate the recombinant proteins to efficiently stimulate humoral and cellular responses, including antigen-specific IgG1 and IgG2a, as well as lymphocyte proliferation. Furthermore, significantly improved protection against T. gondii infection was observed in the mindin group compared with that of Freund's adjuvant and no-adjuvant groups. CONCLUSIONS: The extracellular matrix protein mindin can effectively induce antigen-specific humoral and cell-mediated immune responses. Our study provides a valuable basis for the development of an efficient, safe, non-toxic vaccine adjuvant for future use in humans and animals.
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Antígenos de Protozoos/inmunología , Proteínas de la Matriz Extracelular/inmunología , Proteínas Protozoarias/inmunología , Toxoplasma/inmunología , Toxoplasmosis/inmunología , Adyuvantes Inmunológicos/administración & dosificación , Adyuvantes Inmunológicos/genética , Animales , Anticuerpos Antiprotozoarios/inmunología , Antígenos de Protozoos/administración & dosificación , Antígenos de Protozoos/genética , Proteínas de la Matriz Extracelular/administración & dosificación , Proteínas de la Matriz Extracelular/genética , Femenino , Humanos , Inmunidad Celular , Inmunidad Humoral , Inmunización , Masculino , Ratones , Ratones Endogámicos BALB C , Proteínas Protozoarias/administración & dosificación , Proteínas Protozoarias/genética , Vacunas Antiprotozoos/administración & dosificación , Vacunas Antiprotozoos/genética , Vacunas Antiprotozoos/inmunología , Células TH1/inmunología , Células Th2/inmunología , Toxoplasma/genética , Toxoplasmosis/parasitología , Toxoplasmosis/prevención & control , Vacunas Sintéticas/administración & dosificación , Vacunas Sintéticas/genética , Vacunas Sintéticas/inmunologíaRESUMEN
BACKGROUND: We evaluated the effects of fibronectin, collagen, cadherin, and laminin based extracellular matrix (ECM) protein mimetics coated with mussel derived adhesive protein (MAP) on adhesion and proliferation of chorionic mesenchymal stem cells (cMSCs). METHODS: Human placental chorionic tissues from term third-trimester pregnancies (n=3) were used. The cMSCs were cultured on rationally designed ECM protein mimetics coated with MAP on plastic surfaces with the addition of reduced fetal bovine serum (0.5%, 1% FBS). Adhesion capabilities were monitored by a real time cell analysis system (RTCA) utilizing an impedance method. Proliferation capabilities were monitored by RTCA and MTS assay. RESULTS: Of the ECM protein mimetics tested, GRGDSP(FN) coated surfaces exhibited the highest adhesion and proliferation capabilities on RTCA at FBS concentration of 0.5% and 1%. When 0.5% FBS was added to ECM protein mimetics during the MTS assay, GRGDSP(FN), REDV(FN), and collagen mimetics, GPKGAAGEPGKP(ColI) showed higher cMSCs proliferation compared with the control. When 1% FBS was added, GRGDSP(FN) and TAIPSCPEGTVPLYS(ColIV) showed significant cMSCs proliferation capacity. CONCLUSIONS: Fibronectin mimetics, GRGDSP(FN) amino acid sequence showed the highest adhesion and proliferation capabilities. In addition, results from RTCA assessment of cell viability correlated well with the tetrazolium-based MTS assay.
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Biomimética , Proteínas de la Matriz Extracelular/administración & dosificación , Fibronectinas/administración & dosificación , Células Madre Mesenquimatosas/efectos de los fármacos , Animales , Adhesión Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Corion/citología , Matriz Extracelular/metabolismo , Femenino , Humanos , Células Madre Mesenquimatosas/citología , EmbarazoRESUMEN
Tendons and cartilage are specialized forms of connective tissues originated from common progenitor cells. Initial stages of differentiation of these tissues are characterized by the formation of cell aggregates, which share many molecular markers. Once differentiated, these cells retain considerable plasticity, and chondral metaplasia of tendon and fibrous connective tissues and eventual ossification often accompany degenerative diseases in the adult musculoskeletal system. While this fact is of great relevance for regenerative medicine and aging biology, its molecular basis remains to be elucidated. Gene expression analysis in several physiological and experimental paradigms suggests that differentiation of tendon and cartilage is regulated by a balance in the expression of chondrogenic versus tenogenic genes in the connective tissue cell precursors. Transforming growth factor ß (TGFß) may function both as a profibrogenic or as a prochondrogenic factor for embryonic limb mesoderm and mesenchymal stem cell cultures, but mice that are null for TGFß 2 and 3 lack tendons. Here, we identify ßig-h3 as a factor downstream TGFß signaling regulated by Smad 2 and 3, which is highly expressed in the differentiating tendons and joint capsules. Furthermore, gain- and loss-of-function experiments using limb mesoderm micromass cultures show that ßig-h3 downregulates the expression of cartilage master genes, including Sox9, type II collagen, and Hif-1α. Positive regulation of Sox9 and type II Collagen observed in micromass cultures grown under hypoxic conditions is prevented by exogenous administration of ßIG-H3, and the antichondrogenic influence of ßIG-H3 is lost after Hif-1α silencing with shRNA. Collectively, our findings indicate that ßig-h3 promotes the fibrogenic influence of TGFß signaling, neutralizing the prochondrogenic influence of the hypoxic-inducible factor 1 activated by the hypoxic microenvironment characteristic of limb mesenchymal aggregates.
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Condrocitos/citología , Condrogénesis/fisiología , Tejido Conectivo/crecimiento & desarrollo , Proteínas de la Matriz Extracelular/administración & dosificación , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/fisiología , Factor de Crecimiento Transformador beta/administración & dosificación , Animales , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Embrión de Pollo , Condrocitos/efectos de los fármacos , Condrocitos/fisiología , Condrogénesis/efectos de los fármacos , Tejido Conectivo/anatomía & histología , Tejido Conectivo/efectos de los fármacos , Fibrinolíticos/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/fisiología , Células Madre Mesenquimatosas/efectos de los fármacos , Ratones , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiologíaRESUMEN
Myocardial repair remains a major challenge for both cellular and tissue engineering approaches. Several studies have been conducted looking at utilizing extracellular matrix-based therapies to promote repair after a myocardial infarction. In this review, strategies for treating myocardial infarctions using extracellular matrix-derived peptides are discussed. Using an ischemia/reperfusion myocardial infarction rodent model, we showed that extracellular-matrix-derived peptides were able to induce angiogenesis and alter the negative remodeling seen after a myocardial infarction. This therapy opens up a potentially new non-invasive strategy for repairing damaged cardiac tissue.
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Infarto del Miocardio/terapia , Neovascularización Fisiológica , Ingeniería de Tejidos/métodos , Inductores de la Angiogénesis/administración & dosificación , Animales , Modelos Animales de Enfermedad , Matriz Extracelular/metabolismo , Proteínas de la Matriz Extracelular/administración & dosificación , Humanos , Infarto del Miocardio/fisiopatología , Daño por Reperfusión Miocárdica/fisiopatología , Remodelación VentricularRESUMEN
OBJECTIVE: To determine the effect of topically applied amelogenin extracellular matrix protein(AEMP) in patients with non-healing venous leg ulcers combined with atrophie blanche. METHOD: This retrospective case series of patients with non-healing venous leg ulcers with atrophie blanche of the distal proportion of their lower legs, where non-healing was defined as no progress toward healing for 3 months previously, under standard therapy. Patient records were reviewed for associated diseases, wound diagnoses, distal blood pressure, previous treatments and changes in wound area. Patients were treated with AEMP once a week, for a period of 12 weeks, or until full healing. RESULTS: Eleven patient records were reviewed retrospectively. The median age of the patients was 81 years (range 40-95 years), with a mean wound size of 4.7 ± 3.Scm2 and median wound duration of 6 months (range 3-444 months).AII patients had venous or combined arterial/venous insufficiency. After 12 weeks' treatment with AEMP, complete healing, defined as I 00% re-epithelialisation, was documented in four patients (36%), marked improvement(> SO% epithelialisation) in three patients (54%, 55% and 83% wound closure, respectively), slight improvement in one patient (9.4% wound closure), no change for two patients and worsening in one.AEMP was well tolerated, and no patients reported side effects. CONCLUSION: The results of this retrospective study suggest that AEMP improves healing in chronic venous leg ulcers combined with atrophie blanche.
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Amelogenina/uso terapéutico , Proteínas de la Matriz Extracelular/uso terapéutico , Piel/patología , Úlcera Varicosa/patología , Úlcera Varicosa/fisiopatología , Administración Cutánea , Anciano , Anciano de 80 o más Años , Amelogenina/administración & dosificación , Proteínas de la Matriz Extracelular/administración & dosificación , Femenino , Humanos , Masculino , Estudios Retrospectivos , Úlcera Varicosa/complicaciones , Insuficiencia Venosa/complicacionesRESUMEN
PURPOSE: Opticin is an extracellular matrix glycoprotein that the authors discovered in the vitreous humor of the eye. It is synthesized by the nonpigmented ciliary epithelium and secreted into the vitreous cavity and, unusually for an extracellular matrix molecule, high-level synthesis is maintained into adult life. Here the authors investigated the hypothesis that opticin influences vascular development in the posterior segment of the eye and pathologic angiogenesis into the normally avascular, mature (secondary) vitreous. METHODS: Opticin was localized in murine eyes by immunohistochemistry. An opticin knockout mouse was established and vascular development was compared between knockout and wild-type mice. Wild-type and opticin null mice were compared in the oxygen-induced retinopathy model, a model of pathologic angiogenesis, and this model was also used to assess the effects of intravitreal injection of recombinant opticin into eyes of wild-type mice. RESULTS: Opticin colocalizes with the collagen type II-rich fibrillar network of the vitreous, the inner limiting lamina, the lens capsule, the trabecular meshwork, and the iris. Analyses of the hyaloid and retinal vasculature showed that opticin has no effect on hyaloid vascular regression or developmental retinal vascularization. However, using the oxygen-induced retinopathy model, the authors demonstrated that opticin knockout mice produce significantly more preretinal neovascularization than wild-type mice, and the intravitreal delivery of excess opticin inhibited the formation of neovessels in wild-type mice. CONCLUSIONS: A lack of opticin does not influence vascular development, but opticin is antiangiogenic and inhibits preretinal neovascularization.
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Modelos Animales de Enfermedad , Proteínas de la Matriz Extracelular/administración & dosificación , Proteoglicanos/administración & dosificación , Neovascularización Retiniana/prevención & control , Cuerpo Vítreo/química , Animales , Animales Recién Nacidos , Western Blotting , Colágeno Tipo II/metabolismo , Proteínas de la Matriz Extracelular/genética , Técnica del Anticuerpo Fluorescente Indirecta , Técnicas para Inmunoenzimas , Inyecciones Intravítreas , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Oxígeno/toxicidad , Fragmentos de Péptidos , Proteoglicanos/genética , Proteínas Recombinantes/administración & dosificación , Neovascularización Retiniana/etiología , Neovascularización Retiniana/patología , Cuerpo Vítreo/metabolismoRESUMEN
INTRODUCTION: Intervertebral disc (IVD) degeneration is associated with proteolytic degradation of the extracellular matrix, and its repair requires both the production of extracellular matrix and the downregulation of proteinase activity. These properties are associated with several growth factors. However, the use of growth factors in clinical practice is limited by their high cost. This cost can be circumvented using synthetic peptides, such as Link N, which can stimulate the synthesis of proteoglycan and collagen by IVD cells in vitro. The purpose of the present study was to evaluate the effect of Link N in vivo in a rabbit model of IVD degeneration. METHODS: New Zealand white rabbits received annular puncture in two lumbar discs. Two weeks after puncture, both punctured discs of each rabbit were injected with either Link N or saline. After 2 weeks, nine rabbits were euthanized and the annulus fibrosus (AF) and nucleus pulposus (NP) of Link N-injected and saline-injected IVDs were removed and used to prepare total RNA. Following reverse transcription, quantitative PCR was performed for aggrecan, COL2A1, COL1A1, ADAMTS-4, ADAMTS-5 and MMP-3. After 12 weeks, 19 rabbits were euthanized and the injected IVDs were removed for biochemical and histological analysis. Proteinase K digests were analyzed for DNA and sulfated glycosaminoglycan content. Disc height was monitored radiographically biweekly. RESULTS: Following needle puncture, disc height decreased by about 25% over 2 weeks, and was partially restored by Link N injection. Puncture of the IVD resulted in a trend towards decreased proteoglycan content in both the NP and AF, and a trend towards partial restoration following Link N injection, although under the time course used this did not achieve statistical significance. Link N did not alter the DNA content of the discs. Link N injection led to a significant increase in aggrecan gene expression and a significant decrease in proteinase gene expression in both the NP and AF, when compared with saline alone. CONCLUSIONS: When administered to the degenerate disc in vivo, Link N stimulated aggrecan gene expression and downregulated metalloproteinase expression, and there was a trend towards increased proteoglycan content of the disc, in both the NP and AF. These are features needed for any agent designed to stimulate disc repair. In principle, therefore, Link N supplementation could be an option for treating disc degeneration.