RESUMO
In recent years, increasing threats of radiation exposure and nuclear disasters have become a significant concern for the United States and countries worldwide. Exposure to high doses of radiation triggers a number of potentially lethal effects. Among the most severe is the gastrointestinal (GI) toxicity syndrome caused by the destruction of the intestinal barrier, resulting in bacterial translocation, systemic bacteremia, sepsis, and death. The lack of effective radioprotective agents capable of mitigating radiation-induced damage has prompted a search for novel countermeasures that can mitigate the effects of radiation post exposure, accelerate tissue repair in radiation-exposed individuals, and prevent mortality. We report that a single injection of regenerative peptide TP508 (rusalatide acetate, Chrysalin) 24 h after lethal radiation exposure (9 Gy, LD100/15) appears to significantly increase survival and delay mortality by mitigating radiation-induced intestinal and colonic toxicity. TP508 treatment post exposure prevents the disintegration of GI crypts, stimulates the expression of adherens junction protein E-cadherin, activates crypt cell proliferation, and decreases apoptosis. TP508 post-exposure treatment also upregulates the expression of DCLK1 and LGR5 markers of stem cells that have been shown to be responsible for maintaining and regenerating intestinal crypts. Thus, TP508 appears to mitigate the effects of GI toxicity by activating radioresistant stem cells and increasing the stemness potential of crypts to maintain and restore intestinal integrity. These results suggest that TP508 may be an effective emergency nuclear countermeasure that could be delivered within 24 h post exposure to increase survival and delay mortality, giving victims time to reach clinical sites for advanced medical treatment.
Assuntos
Trato Gastrointestinal/efeitos da radiação , Fragmentos de Peptídeos/farmacologia , Células-Tronco/efeitos dos fármacos , Trombina/farmacologia , Junções Aderentes/efeitos dos fármacos , Junções Aderentes/metabolismo , Animais , Proliferação de Células/efeitos dos fármacos , Trato Gastrointestinal/patologia , Masculino , Camundongos , Camundongos Endogâmicos ICR , Células-Tronco/citologia , Análise de SobrevidaRESUMO
Revascularization of chronic wounds and ischemic tissue is attenuated by endothelial dysfunction and the inability of angiogenic factors to stimulate angiogenesis. We recently showed that TP508, a nonproteolytic thrombin peptide, increases perfusion and NO-dependent vasodilation in hearts with chronic ischemia and stimulates NO production by endothelial cells. In this study, we investigated systemic in vivo effects of TP508 on VEGF-stimulated angiogenesis in vitro using aortic explants in normoxic and hypoxic conditions. Mice were injected with saline or TP508 and 24 h later aortas were removed and cultured to quantify endothelial sprouting. TP508 injection increased endothelial sprouting and potentiated the in vitro response to VEGF. Exposure of control explants to hypoxia inhibited basal and VEGF-stimulated endothelial cell sprouting. This effect of hypoxia was significantly prevented by TP508 injection. Thus, TP508 systemic administration increases responsiveness of aortic endothelial cells to VEGF and diminishes the effect of chronic hypoxia on endothelial cell sprouting. Studies using human endothelial cells in culture suggest that protective effects of TP508 during hypoxia may involve stimulation of endothelial cell NO production. These data suggest potential clinical benefit of using a combination of systemic TP508 and local VEGF as a therapy for revascularization of ischemic tissue.
Assuntos
Indutores da Angiogênese , Hipóxia Celular/efeitos dos fármacos , Fragmentos de Peptídeos/administração & dosagem , Trombina/administração & dosagem , Fator A de Crescimento do Endotélio Vascular/farmacologia , Sequência de Aminoácidos , Animais , Aorta Torácica/citologia , Aorta Torácica/fisiologia , Sinergismo Farmacológico , Quimioterapia Combinada , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/fisiologia , Injeções Intravenosas , Masculino , Camundongos , Camundongos Endogâmicos ICR , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Trombina/química , Técnicas de Cultura de TecidosRESUMO
TP508, a 23-amino-acid peptide representing a portion of human thrombin, promotes tissue revascularization and repair. The molecular mechanisms of TP508 action, however, remain unclear. Nitric oxide (NO) plays a crucial role in regulation of angiogenesis and wound healing. We, therefore, investigated TP508 effects on NO production in human endothelial cells. TP508 stimulated a rapid, dose-dependent, 2- to 4-fold increase in NO production. TP508 induced NO release as early as 5 min. Continued exposure to TP508 for 1-24 h increased NO concentrations over controls by 100.5 +/- 9.6 and 463.3 +/- 24.2 nM, respectively. These levels of NO release were similar to those produced in response to vascular endothelial growth factor (VEGF). TP508- and VEGF-induced NO production was decreased by inhibitors of PI-3K (LY294002) and Src (PP2). TP508 stimulated early transient phosphorylation of Src and Akt. In contrast to VEGF, TP508 stimulation of NO release was inhibited by PKC inhibitor (Go6976) and was independent of intracellular calcium mobilization. These results demonstrate that TP508 and VEGF stimulate NO production to similar levels but through distinct pathways. This study provides new insights into the initial molecular mechanisms by which TP508 may stimulate diverse cellular effects leading to tissue revascularization and wound healing.
Assuntos
Células Endoteliais/efeitos dos fármacos , Óxido Nítrico/metabolismo , Fragmentos de Peptídeos/farmacologia , Trombina/farmacologia , Animais , Cálcio/metabolismo , Bovinos , Células Cultivadas , Relação Dose-Resposta a Droga , Células Endoteliais/metabolismo , Humanos , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Fosforilação , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fatores de Tempo , Regulação para Cima , Fator A de Crescimento do Endotélio Vascular/metabolismo , Quinases da Família src/antagonistas & inibidores , Quinases da Família src/metabolismoRESUMO
Growth plate chondrocytes are susceptible to apoptosis. Terminally differentiated chondrocytes are deleted via apoptosis, which primes the growth plate to vascular invasion and subsequent bone formation. Whether less differentiated resting zone chondrocytes are subject to the same mechanism that governs the apoptotic pathway of more differentiated growth zone chondrocytes is not known. In our current study, we demonstrated that inorganic phosphate, a key inducer of growth plate chondrocyte apoptosis, also causes apoptosis in resting zone chondrocytes, via a pathway similar to the one in growth zone chondrocytes. Our results demonstrated that the conditions that cause growth plate chondrocyte apoptosis lie in the external environment, instead of the differences in differentiation state.
Assuntos
Apoptose/efeitos dos fármacos , Condrócitos/citologia , Condrócitos/efeitos dos fármacos , Fragmentos de Peptídeos/farmacologia , Fosfatos/farmacologia , Animais , Sobrevivência Celular/efeitos dos fármacos , Fragmentação do DNA/efeitos dos fármacos , Feminino , Humanos , Óxido Nítrico/metabolismo , Ratos , Trombina , ômega-N-Metilarginina/farmacologiaRESUMO
BACKGROUND: Cranial radiotherapy (CRT) is an important part of brain tumor treatment, and although highly effective, survivors suffer from long-term cognitive side effects. In this study we aim to establish late-term imaging markers of CRT-induced brain injury and identify functional markers indicative of cognitive performance. Specifically, we aim to identify changes in executive function, brain metabolism, and neuronal organization. METHODS: Male Sprague Dawley rats were fractionally irradiated at 28 days of age to a total dose of 30 Gy to establish a radiation-induced brain injury model. Animals were trained at 3 months after CRT using the 5-choice serial reaction time task. At 12 months after CRT, animals were evaluated for cognitive and imaging changes, which included positron emission tomography (PET) and magnetic resonance imaging (MRI). RESULTS: Cognitive deficit with signs of neuroinflammation were found at 12 months after CRT in irradiated animals. CRT resulted in significant volumetric changes in 38% of brain regions as well as overall decrease in brain volume and reduced gray matter volume. PET imaging showed higher brain glucose uptake in CRT animals. Using MRI, irradiated brains had an overall decrease in fractional anisotropy, lower global efficiency, increased transitivity, and altered regional connectivity. Cognitive measurements were found to be significantly correlated with six image features that included myelin integrity and local organization of the neural network. CONCLUSIONS: These results demonstrate that CRT leads to late-term morphological changes, reorganization of neural connections, and metabolic dysfunction. The correlation between imaging markers and cognitive deficits can be used to assess late-term side effects of brain tumor treatment and evaluate efficacy of new interventions.
RESUMO
Endothelial dysfunction (ED) is characterized by impaired nitric oxide (NO) signaling, decreased NO-dependent vasodilatation, increased vascular inflammation, and diminished response to angiogenic factors. TP508 (Chrysalin), an angiogenic tissue repair peptide, was tested for potential effects on myocardial revascularization and ED using a porcine model of chronic myocardial ischemia. TP508 increased perfusion in ischemic regions up to16-fold (P < .02) and doubled myocardial wall thickening (P < .02) relative to placebo controls. Ischemic arterioles exhibited impaired NO-mediated vasodilation and diminished NO production. TP508 reversed ischemic effects, increasing NO-mediated vasodilation (P < .05), endothelial nitric oxide synthase (eNOS) expression, and NO production. In human endothelial cells, TP508 stimulated eNOS activation (1.84 +/- 0.2-fold; P < .02), increased NO production (85 +/- 18%; P < .02), and prevented hypoxia-induced eNOS downregulation (P < .01). Thus, TP508 reverses ED both in porcine ischemic hearts and cultured human endothelial cells. These results suggest potential therapeutic benefit of TP508 in myocardial revascularization and treatment of ED-related diseases.
Assuntos
Indutores da Angiogênese/farmacologia , Circulação Coronária/efeitos dos fármacos , Vasos Coronários/efeitos dos fármacos , Endotélio Vascular/efeitos dos fármacos , Isquemia Miocárdica/tratamento farmacológico , Revascularização Miocárdica/métodos , Fragmentos de Peptídeos/farmacologia , Animais , Hipóxia Celular , Células Cultivadas , Doença Crônica , Angiografia Coronária , Vasos Coronários/metabolismo , Vasos Coronários/fisiopatologia , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Ecocardiografia sob Estresse , Endotélio Vascular/metabolismo , Endotélio Vascular/fisiopatologia , Humanos , Isquemia Miocárdica/metabolismo , Isquemia Miocárdica/patologia , Isquemia Miocárdica/fisiopatologia , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Suínos , Porco Miniatura , Trombina , Vasodilatação/efeitos dos fármacos , Vasodilatadores/farmacologiaRESUMO
TP508 is a synthetically derived tissue repair peptide that has previously demonstrated safety and potential efficacy in phase I/II clinical trials for the treatment of diabetic foot ulcers. Recent studies show that a single injection of TP508 administered 24 h after irradiation significantly increases survival and delays mortality in murine models of acute radiation mortality. Thus, TP508 is being developed as a potential nuclear countermeasure. Because of the short plasma half-life of TP508, we hypothesize that increasing the peptide bioavailability would increase TP508 efficacy or reduce the dosage required for therapeutic effects. We, therefore, evaluated the covalent attachment of various sizes of polyethylene glycol to TP508 at either its N-terminus or at an internal cysteine. A size-dependent increase in TP508 plasma half-life due to PEGylation was observed in blood samples from male CD-1 mice using fluorescently labeled TP508 and PEGylated TP508 derivatives. Biological activity of PEGylated TP508 derivatives was evaluated using a combination of biologically relevant assays for wound closure, angiogenesis, and DNA repair. PEG5k-TP508 enhanced wound closure after irradiation and enhanced angiogenic sprouting in murine aortic ring segments relative to equimolar dosages of TP508 without enhancing circulating half-life. PEG30k-TP508 extended the plasma half-life by approximately 19-fold while also showing enhanced biological activity. Intermediate-sized PEGylated TP508 derivatives had enhanced plasma half-life but were not active in vivo. Thus, increased half-life does not necessarily correlate with increased biological activity. Nevertheless, these results identify two candidates, PEG5k-TP508 and PEG30k-TP508, for potential development as second-generation TP508 injectable drugs.
Assuntos
Reparo do DNA/efeitos dos fármacos , Fragmentos de Peptídeos/farmacologia , Protetores contra Radiação/farmacologia , Trombina/farmacologia , Cicatrização/efeitos dos fármacos , Animais , Células Cultivadas , Sistemas de Liberação de Medicamentos , Meia-Vida , Histonas , Humanos , Masculino , Camundongos , Neovascularização Fisiológica/efeitos dos fármacos , Fragmentos de Peptídeos/administração & dosagem , Fragmentos de Peptídeos/química , Polietilenoglicóis/química , Protetores contra Radiação/administração & dosagem , Protetores contra Radiação/química , Trombina/administração & dosagem , Trombina/químicaRESUMO
The thrombin peptide, TP508, also known as Chrysalin (OrthoLogic, Tempe, Arizona), is a twenty-three-amino-acid peptide that represents a portion of the receptor-binding domain of the native human thrombin molecule that has been identified as the binding site for a specific class of receptors on fibroblasts and other cells. Preclinical studies with this peptide have shown that it can accelerate tissue repair in both soft and hard tissues by mechanisms that appear to involve up-regulation of genes that initiate a cascade of healing events. These events include recruitment and activation of inflammatory cells, directed migration of cells (chemotaxis), cell proliferation, elaboration of extra-cellular matrix, and accelerated revascularization of the healing tissues. Early preclinical dermal wound-healing studies showed that TP508 accelerated healing of both incisional wounds and full-thickness excisional wounds in normal and ischemic skin. In all of these studies, the accelerated healing was associated with increased neovascularization across the incision or in the granulating wound bed. Studies in a rat fracture model have also shown that TP508 accelerates the rate of fracture repair. Gene array analysis of fracture callus from control and TP508-treated fractures indicated that TP508 treatment was associated with an up-regulation of early response elements, inflammatory mediators, and genes related to angiogenesis. Similar to what had been seen in dermal wounds, histology from rat fracture callus twenty-one days after treatment indicated that fractures treated with TP508 had significantly more large functional blood vessels than did fractures in the control animals. In vitro studies support these in vivo data and indicate that TP508 may have a direct angiogenic effect by promoting the rate of new vessel growth. The results from phase-1 and phase-2 human clinical studies have shown a positive stimulatory effect of TP508 in the healing of diabetic ulcers and in the repair of fractures to the distal aspect of the radius. Collectively, these studies suggest that TP508 accelerates tissue repair by initiating a cascade of events that lead to an increased rate of tissue revascularization and regeneration.
Assuntos
Neovascularização Fisiológica/efeitos dos fármacos , Fragmentos de Peptídeos/farmacologia , Trombina/farmacologia , Cicatrização/efeitos dos fármacos , Animais , Pé Diabético/tratamento farmacológico , Humanos , Fragmentos de Peptídeos/uso terapêutico , Fraturas do Rádio/tratamento farmacológico , Ratos , Trombina/uso terapêuticoRESUMO
There is increasing evidence that radiation-induced damage to endothelial cells and loss of endothelial function may contribute to both acute radiation syndromes and long-term effects of whole-body nuclear irradiation. Therefore, several drugs are being developed to mitigate the effects of nuclear radiation, most of these drugs will target and protect or regenerate leukocytes and platelets. Our laboratory has demonstrated that TP508, a 23-amino acid thrombin peptide, activates endothelial cells and stem cells to revascularize and regenerate tissues. We now show that TP508 can mitigate radiation-induced damage to endothelial cells in vitro and in vivo. Our in vitro results demonstrate that human endothelial cells irradiation attenuates nitric oxide (NO) signaling, disrupts tube formation and induces DNA double-strand breaks (DSB). TP508 treatment reverses radiation effects on NO signaling, restores tube formation and accelerates the repair of radiation-induced DSB. The radiation-mitigating effects of TP508 on endothelial cells were also seen in CD-1 mice where systemic injection of TP508 stimulated endothelial cell sprouting from aortic explants after 8 Gy irradiation. Systemic doses of TP508 that mitigated radiation-induced endothelial cell damage, also significantly increased survival of CD-1 mice when injected 24 h after 8.5 Gy exposure. These data suggest that increased survival observed with TP508 treatment may be due to its effects on vascular and microvascular endothelial cells. Our study supports the usage of a regenerative drug such as TP508 to activate endothelial cells as a countermeasure for mitigating the effects of nuclear radiation.
Assuntos
Reparo do DNA/efeitos dos fármacos , Reparo do DNA/efeitos da radiação , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/efeitos da radiação , Fragmentos de Peptídeos/farmacologia , Trombina/farmacologia , Sequência de Aminoácidos , Animais , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Quebras de DNA de Cadeia Dupla/efeitos da radiação , Relação Dose-Resposta à Radiação , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Humanos , Masculino , Camundongos , Óxido Nítrico/biossíntese , Análise de SobrevidaRESUMO
This study investigated the in vivo degradation of poly(propylene fumarate) (PPF)/poly(DL-lactic-co-glycolic acid) (PLGA) composite scaffolds designed for controlled release of osteogenic factors. PPF/PLGA composites were implanted into 15.0mm segmental defects in the rabbit radius, harvested after 12 and 18 weeks, and analyzed using histological techniques to assess the extent of polymer degradation as well as the tissue response within the pores of the scaffolds. Polymer degradation was limited to micro-fragmentation of the scaffold at the ends and edges of the implant at both 12 and 18 weeks. The tissue within the pores of the scaffold consisted of fibrous tissue, blood vessels and some inflammatory cells. In areas where polymer breakdown was evident, an increased inflammatory response was observed. In contrast, areas of bone ingrowth into the polymer scaffold were characterized by minimal inflammatory response and polymer degradation. Our results show that minimal degradation of porous PPF occurs within 18 weeks of implantation in a rabbit model. Further, the in vivo degradation data of porous PPF/PLGA scaffolds are comparable with earlier obtained in vitro data.
Assuntos
Fumaratos/química , Ácido Láctico/química , Ácido Poliglicólico/química , Polímeros/química , Polipropilenos/química , Animais , Biodegradação Ambiental , Copolímero de Ácido Poliláctico e Ácido Poliglicólico , Coelhos , Rádio (Anatomia)RESUMO
This study focused on the assessment of radiography, microcomputed tomography, and histology for the evaluation of bone formation in a 15.0-mm defect in the rabbit radius after the implantation of a tissue-engineered construct. Radiography was found to be useful as a noninvasive method for obtaining images of calcified tissue throughout the time course of the experiment. With this method, however, image quality was low, making it difficult to obtain precise information about the location and quantity of the bone formed. Microcomputed tomography was used to create three-dimensional reconstructions of the bone (25-microm resolution). These reconstructions allowed for greater spatial resolution than the radiography, but did not allow for imaging of the implanted scaffold material or the surrounding, nonmineralized tissue. To visualize all materials within the defect area at the cellular level, histology was used. Histological analysis, however, is a destructive technique that did not allow for any further analysis of the samples. Each technique examined here has its own advantages and limitations, but each yields unique information regarding bone regeneration. It is only through the use of all three techniques that complete characterization of the bone growth and tissue/construct responses after implantation in vivo.
Assuntos
Osso e Ossos/diagnóstico por imagem , Osso e Ossos/fisiologia , Técnicas Histológicas/métodos , Radiografia/métodos , Engenharia Tecidual/métodos , Tomografia Computadorizada por Raios X/métodos , Acrilatos/síntese química , Acrilatos/química , Animais , Materiais Biocompatíveis/síntese química , Materiais Biocompatíveis/química , Regeneração Óssea , Osso e Ossos/citologia , Preparações de Ação Retardada/farmacologia , Relação Dose-Resposta a Droga , Portadores de Fármacos , Feminino , Fumaratos/síntese química , Fumaratos/química , Glicolatos/química , Ácido Láctico , Microesferas , Peso Molecular , Tamanho da Partícula , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/farmacologia , Ácido Poliglicólico , Copolímero de Ácido Poliláctico e Ácido Poliglicólico , Polímeros/química , Polipropilenos/síntese química , Polipropilenos/química , Coelhos , Rádio (Anatomia)/citologia , Rádio (Anatomia)/diagnóstico por imagem , Rádio (Anatomia)/fisiologia , Trombina/química , Trombina/farmacologia , Fatores de TempoRESUMO
This research investigates the release of plasmid DNA in vitro from novel, injectable hydrogels based on the polymer oligo(poly(ethylene glycol) fumarate) (OPF). These biodegradable hydrogels can be crosslinked under physiological conditions to physically entrap plasmid DNA. The DNA release kinetics were characterized fluorescently with the PicoGreen and OliGreen Reagents as well as through the use of radiolabeled plasmid. Further, the ability of the released DNA to be expressed was assessed through bacterial transformations. It was found that plasmid DNA can be released in a sustained, linear fashion over the course of 45-62 days, with the release kinetics depending upon the molecular weight of the poly(ethylene glycol) from which the OPF was synthesized. Two formulations of OPF were synthesized from poly(ethylene glycol) of a nominal molecular weight of either 3.35K (termed OPF 3K) or 10K (termed OPF 10K). By the time the gels had completely degraded, 97.8+/-0.3% of the initially loaded DNA was recovered from OPF 3K hydrogels, with 80.8+/-1.9% of the initial DNA retaining its double-stranded form. Likewise, for OPF 10K gels, 92.1+/-4.3% of the initially loaded DNA was recovered upon complete degradation of the gels, with 81.6+/-3.8% of the initial DNA retaining double-stranded form. Experiments suggest that the release of plasmid DNA from OPF hydrogels is dominated by the degradation of the gels. Bacterial transformation results indicated that the DNA retained bioactivity over the course of 42 days of release. Thus, these studies demonstrate the potential of OPF hydrogels in controlled gene delivery applications.
Assuntos
Materiais Biocompatíveis/química , DNA/metabolismo , Hidrogéis/química , Plasmídeos/química , Poliésteres/química , Polietilenoglicóis/química , DNA/química , Técnicas de Transferência de Genes , CinéticaRESUMO
The thrombin-related peptide, TP508, has been shown to promote soft tissue healing and fracture repair. One possible clinical application of TP508 is to accelerate bone regeneration during distraction osteogenesis, which is a lengthy procedure involving significant complications. In this study, we tested the ability of TP508 to accelerate the consolidation phase of distraction osteogenesis in a rabbit model of leg lengthening. Twenty-three rabbits had left tibiae lengthened for 1 cm over a period of 6 days. TP 508 (0, 30 and 300 microg in 300 microl saline) was injected into the distraction gaps at the beginning and the end of the lengthening phase, and all the animals were killed 2 weeks after lengthening. By the end of experiment, more animals in the TP508 treated groups had complete bony union of the distraction gaps when compared to the saline treated group. pQCT examination of the regenerates demonstrated a significantly greater bone mineral density (BMD) in the TP508 treated groups relative to the saline control group, but no statistical difference in the BMD was found between the two dosages of TP508. Bone consolidation and bone remodeling was far advanced in the TP508 300 microg treated group, and the regenerates mainly consisted of well-vascularized woven bone. In contrast, in the group that received the 30 microg TP508 treatment, focal bone defects and discontinuities of the new cortices were evident in some but not all animals. In the saline control group a majority of the animals showed large amounts of fibrous and cartilaginous tissues in the regenerates, and none of the regenerates had completed consolidation. This study has demonstrated that local application of TP508 enhanced bone formation and consolidation during distraction osteogenesis in the rabbit. The findings indicate that TP508 may be useful in promoting osteogenesis in situations when augmentative treatment for bone formation and consolidation are needed.
Assuntos
Osteogênese por Distração , Osteogênese/efeitos dos fármacos , Fragmentos de Peptídeos/farmacologia , Trombina/farmacologia , Animais , Regeneração Óssea/efeitos dos fármacos , Masculino , Neovascularização Fisiológica/efeitos dos fármacos , CoelhosRESUMO
Previous studies have shown that a single injection of thrombin peptide (TP508) accelerates fracture repair in a closed rat femoral fracture model. The present study was conducted to elucidate the molecular mechanisms of TP508 action using Affymetrix genome-scale profiling and to link early gene expression changes to fracture histology and bone strength changes. Treatment of femoral fractures with TP508 accelerated fracture repair as determined by destructive torsion testing. Blinded histological analysis demonstrated that TP508-treated fracture callus had a significant increase in blood vessels relative to the controls. Gene array analysis showed that TP508 significantly induced expression of early growth factors, inflammatory response modifiers, and angiogenesis-related genes. This study therefore suggests that TP508 promotes fracture repair through a mechanism that involves an increased induction of a number of growth factors, enhanced expression of inflammatory mediators, and angiogenesis-related genes.
Assuntos
Consolidação da Fratura/efeitos dos fármacos , Substâncias de Crescimento/genética , Mediadores da Inflamação/metabolismo , Neovascularização Fisiológica , Fragmentos de Peptídeos/farmacologia , Trombina/farmacologia , Animais , Fenômenos Biomecânicos , Regulação da Expressão Gênica , Masculino , Análise de Sequência com Séries de Oligonucleotídeos , Ratos , Ratos Sprague-Dawley , Regulação para CimaRESUMO
This study was designed to assess the influence of varied release kinetics of the osteogenic thrombin peptide TP508 from osteoconductive poly(propylene fumarate)-based (PPF) composite scaffolds on bone formation in vivo. Four classes of scaffolds were constructed with different TP508 dosages (200, 100, or 0 microg) and release kinetics (large burst release, minimal burst release, or no release) and implanted in 15.0 mm segmental defects in rabbit radii. The animals were euthanized at 12 weeks and the implants were analyzed by light microscopy, histological scoring analysis, and histomorphometric analysis. Samples from all classes displayed bone growth within the pores of the scaffold near the edges of the defect. In areas where bone was not observed, the pores were filled with mostly fibrous tissue and exhibited minimal inflammatory response for all classes. In contrast to other scaffold classes, scaffolds containing a total dose of 200 microg TP508 and exhibiting a large burst release profile showed statistically more bone formation guided along the surface of the scaffold, with these scaffolds averaging 80% of the defect length bridged with bone compared to 10% or less bridged for the other scaffold classes. These results demonstrate that the extent of in vivo bone formation in response to controlled release from PPF-composite scaffolds is determined by the release kinetics of the incorporated osteogenic peptide.
Assuntos
Implantes Absorvíveis , Fumaratos/uso terapêutico , Osteogênese/efeitos dos fármacos , Fragmentos de Peptídeos/farmacologia , Polipropilenos/uso terapêutico , Trombina/farmacologia , Animais , Relação Dose-Resposta a Droga , Implantes Experimentais , Cinética , Fragmentos de Peptídeos/administração & dosagem , Coelhos , Trombina/administração & dosagem , Engenharia Tecidual/métodosRESUMO
The thrombin peptide, TP508, accelerates tissue repair and initiates a cascade of cellular events. We have previously shown that alpha-thrombin induces cytokine expression in human mononuclear cells. We, therefore, investigated the possibility that TP508 might activate cytokine production and intracellular signaling pathways associated with cytokine activation. Our results show that TP508 induces cytokine expression in human mononuclear cells. TP508 treatment enhances extracellular signal-regulated kinase (Erk1/2) activities in U937 cells, as well as Erk1/2 and p38 activation in Jurkat T cells. These data support the hypothesis that TP508 may accelerate tissue repair through the activation of the inflammatory response.
Assuntos
Citocinas/metabolismo , Peptídeos/farmacologia , Transdução de Sinais/efeitos dos fármacos , Trombina/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Ativação Enzimática/efeitos dos fármacos , Ensaio de Imunoadsorção Enzimática , Humanos , Immunoblotting , Interleucina-1/metabolismo , Interleucina-2/metabolismo , Células Jurkat , Cinética , Leucócitos Mononucleares/efeitos dos fármacos , Lipopolissacarídeos/farmacologia , Proteína Quinase 1 Ativada por Mitógeno/efeitos dos fármacos , Proteína Quinase 3 Ativada por Mitógeno/efeitos dos fármacos , Oligonucleotídeos Antissenso/farmacologia , Peptídeos/química , Fosforilação , Fito-Hemaglutininas/farmacologia , Trombina/química , Células U937 , Proteínas Quinases p38 Ativadas por Mitógeno/efeitos dos fármacosRESUMO
Poly(D,L-lactic-co-glycolic acid)/poly(ethylene glycol) (PLGA/PEG) blend microparticles loaded with the osteogenic peptide TP508 were added to a mixture of poly(propylene fumarate) (PPF), poly(propylene fumarate)-diacrylate (PPF-DA), and sodium chloride (NaCl) for the fabrication of PPF composite scaffolds that could allow for tissue ingrowth as well as for the controlled release of TP508 when implanted in an orthopedic defect site. In this study, PPF composites were fabricated and the in vitro release kinetics of TP508 were determined. TP508 loading within the PLGA/PEG microparticles, PEG content within the PLGA/PEG microparticles, the microparticle content of the PPF composite polymer component, and the leachable porogen initial mass percent of the PPF composites were varied according to a fractional factorial design and the effect of each variable on the release kinetics was determined for up to 28 days. Each composite formulation released TP508 with a unique release profile. The initial release (release through day 1) of the PLGA/PEG microparticles was reduced upon inclusion in the PPF composite formulations. Day 1 normalized cumulative mass release from PPF composites ranged from 0.14+/-0.01 to 0.41+/-0.01, whereas the release from PLGA/PEG microparticles ranged from 0.31+/-0.02 to 0.58+/-0.01. After 28 days, PPF composites released 53+/-4% to 86+/-2% of the entrapped peptide resulting in cumulative mass releases ranging from 0.14+/-0.01 microg TP508/mm(3) scaffold to 2.46+/-0.05 microg TP508/mm(3) scaffold. The results presented here demonstrate that PPF composites can be used for the controlled release of TP508 and that alterations in the composite's composition can lead to modulation of the TP508 release kinetics. These composites can be used to explore the effects varied release kinetics and dosages on the formation of bone in vivo.
Assuntos
Implantes Absorvíveis , Cimentos Ósseos/química , Fumaratos/química , Fragmentos de Peptídeos/química , Polietilenoglicóis/química , Poliglactina 910/química , Polipropilenos/química , Trombina/química , Preparações de Ação Retardada/química , Injeções , Cinética , Microesferas , Tamanho da Partícula , PorosidadeRESUMO
The synthetic peptide, TP508 (Chrysalin), was delivered to rabbit segmental bone defects in biodegradable controlled-release PLGA microspheres to determine its potential efficacy for enhancing healing of non-critically and critically sized segmental defects. Non-critically sized radial defects were created in the forelimbs of New Zealand White rabbits, which were randomized into three treatment groups receiving 10, 50 and 100 microg doses of TP508 in the right radius and control microspheres (without TP508) in the left radius. Torsional testing of the radii at six weeks showed a significant increase in ultimate torque, failure torque, ultimate energy, failure energy, and stiffness when treated with TP508 compared to controls (p<0.01 for all measures). Thus, TP508 appeared to enhance or accelerate bone growth in these defects. In a second set of experiments, critically sized ulnar defects were created in the forelimbs of New Zealand White rabbits, which were randomized into two groups with each rabbit receiving microspheres with 100 or 200 microg of TP508 into the right ulnar defect and control microspheres (without TP508) alone into the left ulnar defect. Bone healing was evaluated with plain radiographs, synchrotron-based microtomography, and mechanical testing. Radiographs of the rabbit limbs scored by three blinded, independent reviewers demonstrated a significantly higher degree of healing when treated with TP508 than their untreated control limbs (p<0.05). Three-dimensional synchrotron tomography of a limited number of samples showed that the new bone in TP508-treated samples had a less porous surface appearance and open marrow spaces, suggesting progression of bone remodeling. Torsional testing of the ulnae at nine weeks showed a significant increase in maximum torque and failure energy when treated with TP508 compared to controls (p<0.01 for both measures). These results suggest that TP508 in a controlled release delivery vehicle has the potential to enhance healing of segmental defects in both critically and non-critically sized defects.
Assuntos
Regeneração Óssea/efeitos dos fármacos , Fragmentos de Peptídeos/farmacologia , Trombina/farmacologia , Cicatrização/efeitos dos fármacos , Animais , Fenômenos Biomecânicos , Masculino , Microesferas , Fragmentos de Peptídeos/administração & dosagem , Coelhos , Radiografia , Rádio (Anatomia)/diagnóstico por imagem , Trombina/administração & dosagem , Ulna/diagnóstico por imagemRESUMO
Coronary artery disease results in progressive vascular stenosis associated with chronic myocardial ischemia. Vascular endothelial growth factor (VEGF) stimulates endothelial cell angiogenic responses to revascularize ischemic tissues; however, the effect of chronic hypoxia on the responsiveness of endothelial cells to VEGF remains unclear. We, therefore, investigated whether hypoxia alters VEGF-stimulated signaling and angiogenic responses in primary human coronary artery endothelial (HCAE) cells. Exposure of HCAE cells to hypoxia (1% O(2)) for 24 h decreased VEGF-stimulated endothelial cell migration ( approximately 82%), proliferation ( approximately 30%), and tube formation. Hypoxia attenuated VEGF-stimulated activation of endothelial nitric oxide (NO) synthase (eNOS) ( approximately 72%) and reduced NO production in VEGF-stimulated cells from 237 +/- 38.8 to 61.3 +/- 28.4 nmol/l. Moreover, hypoxia also decreased the ratio of phosphorylated eNOS to total eNOS in VEGF-stimulated cells by approximately 50%. This effect was not observed in thrombin-stimulated cells, suggesting that hypoxia specifically inhibited VEGF signaling upstream of eNOS phosphorylation. VEGF-induced activation of Akt, ERK1/2, p38, p70S6 kinases, and S6 ribosomal protein was also attenuated in hypoxic cells. Moreover, VEGF-stimulated phosphorylation of VEGF receptor-2 (KDR) at Y996 and Y1175 was decreased by hypoxia. This decrease correlated with a 70 +/- 12% decrease in KDR protein expression. Analysis of mRNA from these cells showed that hypoxia reduced steady-state levels of KDR mRNA by 52 +/- 16% and decreased mRNA stability relative to normoxic cells. Our findings demonstrate that chronic hypoxia attenuates VEGF-stimulated signaling in HCAE cells by specific downregulation of KDR expression. These data provide a novel explanation for the impaired angiogenic responses to VEGF in endothelial cells exposed to chronic hypoxia.
Assuntos
Células Endoteliais/metabolismo , Hipóxia/metabolismo , Isquemia Miocárdica/metabolismo , Neovascularização Fisiológica/fisiologia , Fator A de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Adulto , Capilares/citologia , Capilares/metabolismo , Divisão Celular/fisiologia , Movimento Celular/fisiologia , Células Cultivadas , Doença Crônica , Vasos Coronários/citologia , Regulação para Baixo/fisiologia , Células Endoteliais/citologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Expressão Gênica/fisiologia , Humanos , Hipóxia/patologia , Isquemia Miocárdica/patologia , Óxido Nítrico Sintase Tipo III/genética , Óxido Nítrico Sintase Tipo III/metabolismo , Fosforilação/fisiologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Transdução de Sinais/fisiologia , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Rusalatide acetate (Chrysalin) is an investigational drug being evaluated for treatment of chronic wounds and fractures. Rusalatide acetate interacts with cell surface receptors to stimulate a cascade of cellular and molecular wound healing events, including activation of nitric oxide signaling. Rusalatide acetate significantly accelerated healing of diabetic foot ulcers and distal radius fractures in Phase I/II clinical trials. Subsequently, in one of the largest Phase III fracture studies to date, rusalatide acetate showed significant acceleration of distal radius fracture healing radiographically but failed to meet its primary clinical endpoint - time to removal of immobilization - within the intent-to-treat population. Subset analysis showed that rusalatide acetate met this primary clinical endpoint and significantly accelerated radiographic healing in osteopenic women. Rusalatide acetate may therefore show its greatest efficacy in healing-impaired patients.