RESUMO
MS-based proteomics has been the method of choice for biomarker discovery in the field of traumatic brain injury (TBI). Due to its high sensitivity and specificity, MS is now being explored for biomarker quantitative validation in tissue and biofluids. In this study, we demonstrate the use of MS in both qualitative protein identification and targeted detection of acute TBI biomarkers released from degenerating cultured rat cortical mixed neuronal cells, mimicking intracellular fluid in the central nervous system after TBI. Calpain activation was induced by cell treatment with maitotoxin (MTX), a known calcium channel opener. Separate plates of mixed neuronal-glial culture were subjected to excitotoxin N-methyl-D-aspartate (NMDA) and apoptotic inducer staurosporine. Acute TBI biomarkers, GFAP and UCH-L1, were first detected and assessed in the culture media by Western blot. The cell-conditioned media were then trypsinized and subjected to bottom up proteomic analysis. GFAP was readily detected by data-dependent scanning but not UCH-L1. As a proof-of-principle study, rat glia-enriched cell cultures treated with MTX were used to investigate the time-dependent release of GFAP breakdown product by Western blot and for isotope dilution MS absolute quantitation method development. Absolute quantitation of the GFAP release was conducted using the three cortical mixed neuronal cell cultures treated with different agents. Other differentially expressed proteins identified in the glial-enriched and cortical mixed neuronal cell culture models were further analyzed by bioinformatic tools. In summary, this study demonstrates the use of MS in both protein identification and targeted quantitation of acute TBI biomarkers and is the preliminary step toward development of TBI biomarker validation by targeted MS.
Assuntos
Lesões Encefálicas/metabolismo , Córtex Cerebral/metabolismo , Espectrometria de Massas/métodos , Neuroglia/metabolismo , Neurônios/metabolismo , Proteômica/métodos , Animais , Apoptose/efeitos dos fármacos , Biomarcadores/análise , Biomarcadores/metabolismo , Lesões Encefálicas/patologia , Células Cultivadas , Córtex Cerebral/química , Córtex Cerebral/citologia , Modelos Animais de Doenças , Proteína Glial Fibrilar Ácida/análise , Proteína Glial Fibrilar Ácida/química , Proteína Glial Fibrilar Ácida/metabolismo , Toxinas Marinhas/farmacologia , N-Metilaspartato/farmacologia , Necrose/metabolismo , Neuroglia/química , Neuroglia/citologia , Neurônios/química , Neurônios/citologia , Oxocinas/farmacologia , Mapas de Interação de Proteínas/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacos , Estaurosporina/farmacologia , Ubiquitina Tiolesterase/análise , Ubiquitina Tiolesterase/metabolismoRESUMO
Protein tyrosine nitration is a post-translational modification commonly used as a marker of cellular oxidative stress associated with numerous pathophysiological conditions. We focused on ubiquitin carboxyl terminal hydrolase-L1 (UCH-L1) and glyceraldehyde-3-phosphate (GAPDH) which are high-abundant brain proteins that have been identified to be highly susceptible to oxidative modification. Both UCH-L1 and GAPDH have been linked to the pathogenesis of Alzheimer's and Parkinson's disease, however specific nitration sites have not been elucidated. Identification of specific nitration sites and quantitation of endogenous nitrated proteins are important in correlating this modification to disease pathology. In this study, purified UCH-L1 and GAPDH were nitrated in vitro with peroxynitrite and the presence of nitrated proteins was confirmed by anti-3-nitrotyrosine Western blots. Data-dependent LC-MS/MS analysis identified several distinct tyrosine nitration sites in UCH-L1 (Tyr-80) and GAPDH (Tyr-47, Tyr-92, and Tyr-312). Subsequent validation with synthetic peptides was conducted for selected nitropeptides. An LC-MS/MS method was developed for semi-quantitative determination of the synthetic nitropeptides: KGQEVSPKVY(*) (UCH-L1) and mFQY(*) DSTHGKF (GAPDH). The nitropeptides were detectable in the mid-attomole range and the peak area response was linear over three orders of magnitude. Targeted analysis of endogenous UCH-L1 and GAPDH nitration was then conducted in an in vivo second-hand smoke rat model to evaluate the utility of this approach.
Assuntos
Gliceraldeído-3-Fosfato Desidrogenases/química , Estresse Oxidativo/fisiologia , Poluição por Fumaça de Tabaco , Tirosina/análogos & derivados , Ubiquitina Tiolesterase/química , Sequência de Aminoácidos , Animais , Encéfalo/metabolismo , Química Encefálica , Gliceraldeído-3-Fosfato Desidrogenases/metabolismo , Humanos , Immunoblotting , Masculino , Dados de Sequência Molecular , Nitrosação , Oxirredução , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Coelhos , Ratos , Ratos Sprague-Dawley , Reprodutibilidade dos Testes , Tirosina/análise , Tirosina/química , Tirosina/metabolismo , Ubiquitina Tiolesterase/metabolismoRESUMO
In order to explore early events during the association of plasminogen activator inhibitor-1 (PAI-1) with its cofactor vitronectin, we have applied a robust strategy that combines protein engineering, fluorescence spectroscopy, and rapid reaction kinetics. Fluorescence stopped-flow experiments designed to monitor the rapid association of PAI-1 with vitronectin indicate a fast, concentration-dependent, biphasic binding of PAI-1 to native vitronectin but only a monophasic association with the somatomedin B (SMB) domain, suggesting that multiple phases of the binding interaction occur only when full-length vitronectin is present. Nonetheless, in all cases, the initial fast interaction is followed by slower fluorescence changes attributed to a conformational change in PAI-1. Complementary experiments using an engineered, fluorescently silent PAI-1 with non-natural amino acids showed that concomitant structural changes occur as well in native vitronectin. Furthermore, we have measured the effect of vitronectin on the rate of insertion of the reactive center loop into beta-sheet A of PAI-1 during reaction with target proteases. With a variety of PAI-1 variants, we observe that both full-length vitronectin and the SMB domain have protease-specific effects on the rate of loop insertion but that the two exhibit clearly different effects. These results support a model for PAI-1 binding to vitronectin in which the interaction surface extends beyond the region of PAI-1 occupied by the SMB domain. In support of this model are recent results that define a PAI-1-binding site on vitronectin that lies outside the somatomedin B domain (Schar, C. R., Blouse, G. E., Minor, K. H., and Peterson, C. B. (2008) J. Biol. Chem. 283, 10297-10309) and the complementary site on PAI-1 (Schar, C. R., Jensen, J. K., Christensen, A., Blouse, G. E., Andreasen, P. A., and Peterson, C. B. (2008) J. Biol. Chem. 283, 28487-28496).
Assuntos
Inibidor 1 de Ativador de Plasminogênio/química , Inibidor 1 de Ativador de Plasminogênio/metabolismo , Vitronectina/química , Vitronectina/metabolismo , Sítios de Ligação , Fluorescência , Corantes Fluorescentes/metabolismo , Humanos , Cinética , Modelos Moleculares , Peptídeo Hidrolases/metabolismo , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Propriedades de Superfície , Triptofano/metabolismo , Vitronectina/sangueRESUMO
Calpastatin, a naturally occurring protein, is the only inhibitor that is specific for calpain. A novel blood-brain barrier (BBB)-permeant calpastatin-based calpain inhibitor, named B27-HYD, was developed and used to assess calpain's contribution to neurological dysfunction after stroke in rats. Postischemic administration of B27-HYD reduced infarct volume and neurological deficits by 35% and 44%, respectively, compared to untreated animals. We also show that the pharmacologic intervention has engaged the intended biologic target. Our data further demonstrates the potential utility of SBDP145, a signature biomarker of acute brain injury, in evaluating possible mechanisms of calpain in the pathogenesis of stroke and as an adjunct in guiding therapeutic decision making.
Assuntos
Encéfalo/efeitos dos fármacos , Calpaína/uso terapêutico , Infarto Cerebral/tratamento farmacológico , Inibidores de Cisteína Proteinase/uso terapêutico , Fragmentos de Peptídeos/uso terapêutico , Animais , Barreira Hematoencefálica/metabolismo , Encéfalo/fisiopatologia , Proteínas de Ligação ao Cálcio/administração & dosagem , Proteínas de Ligação ao Cálcio/uso terapêutico , Calpaína/administração & dosagem , Calpaína/antagonistas & inibidores , Calpaína/metabolismo , Infarto Cerebral/fisiopatologia , Inibidores de Cisteína Proteinase/administração & dosagem , Inibidores de Cisteína Proteinase/metabolismo , Modelos Animais de Doenças , Masculino , Fragmentos de Peptídeos/administração & dosagem , Fragmentos de Peptídeos/metabolismo , Ratos , Ratos Wistar , Espectrina/metabolismoRESUMO
The effects of selective inhibition of cathepsins B and L on postischemic protein alterations in the brain were investigated in a rat model of middle cerebral artery occlusion (MCAO). Cathepsin B activity increased predominantly in the subcortical region of the ischemic hemisphere where the levels of collapsing mediator response protein 2, heat shock cognate 70 kDa protein, 60 kDa heat shock protein, protein disulfide isomerase A3 and albumin, were found to be significantly elevated. Postischemic treatment with Cbz-Phe-Ser(OBzl)-CHN(2), cysteine protease inhibitor 1 (CP-1), reduced infarct volume, neurological deficits and cathepsin B activity as well as the amount of heat shock proteins and albumin found in the brain. Our data strongly suggests that the decrease in heat shock protein levels and the significant reduction of serum albumin leakage into the brain following acute treatment with CP-1 is indicative of less secondary ischemic damage, which ultimately, is related to less cerebral tissue loss and improved neurological recovery of the animals.
Assuntos
Isquemia Encefálica/metabolismo , Catepsina B/antagonistas & inibidores , Catepsina B/metabolismo , Catepsinas/antagonistas & inibidores , Catepsinas/metabolismo , Cisteína Endopeptidases/metabolismo , Proteínas de Choque Térmico/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Adaptação Fisiológica , Animais , Catepsina L , Masculino , Ratos , Ratos WistarRESUMO
It is well documented that tumor suppressive maspin inhibits tumor cell invasion and extracellular matrix remodeling. Maspin is a cytosolic, cell surface-associated, and secreted protein in the serine protease inhibitor superfamily. Although several molecules have been identified as candidate intracellular maspin targets, the extracellular maspin target(s) remains elusive. Although maspin does not directly inhibit urokinase-type plasminogen activator (uPA) activity, we have shown evidence that maspin may block the pericellular proteolysis mediated by cell surface-associated uPA. In the current study, maspin significantly inhibited the Ca2+ reduction-induced detachment of DU145 cells. This maspin effect was associated with increased and sustained levels of mature focal adhesion contacts (FAC). We noted that maspin (a) colocalized with uPA and uPA receptor (uPAR), (b) enhanced the interaction between uPAR and low-density lipoprotein receptor related protein, and (c) induced rapid internalization of uPA and uPAR. The maspin effects on surface-associated uPA and uPAR required the interaction between uPA and uPAR. Further biochemical and biophysical analyses revealed that maspin specifically bound to pro-uPA with a deduced K(d) of 270 nmol/L and inhibited the plasmin-mediated pro-uPA cleavage. Interestingly, substitution of maspin p1' site Arg340 in the reactive site loop (RSL) with alanine not only abolished the binding to pro-uPA but also diminished the maspin effects on pro-uPA cleavage and cell detachment. These data show an important role of maspin RSL in regulating the uPA/uPAR-dependent cell detachment. Together, our data led to a new hypothesis that maspin may stabilize mature FACs by quenching localized uPA/uPAR complex before uPA activation.
Assuntos
Genes Supressores de Tumor/fisiologia , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Receptores de Superfície Celular/metabolismo , Serpinas/fisiologia , Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Adesão Celular/efeitos dos fármacos , Adesão Celular/fisiologia , Linhagem Celular Tumoral , Fibrinolisina/antagonistas & inibidores , Fibrinolisina/metabolismo , Adesões Focais/efeitos dos fármacos , Adesões Focais/fisiologia , Humanos , Masculino , Neoplasias da Próstata/enzimologia , Neoplasias da Próstata/genética , Receptores de Ativador de Plasminogênio Tipo Uroquinase , Proteínas Recombinantes/metabolismo , Serpinas/genética , Serpinas/metabolismo , Serpinas/farmacologia , TransfecçãoRESUMO
Traumatic brain injury (TBI) represents a critical health problem of which diagnosis, management, and treatment remain challenging. TBI is a contributing factor in approximately one-third of all injury-related deaths in the United States. The Centers for Disease Control and Prevention estimate that 1.7 million people suffer a TBI in the United States annually. Efforts continue to focus on elucidating the complex molecular mechanisms underlying TBI pathophysiology and defining sensitive and specific biomarkers that can aid in improving patient management and care. Recently, the area of neuroproteomics-systems biology is proving to be a prominent tool in biomarker discovery for central nervous system injury and other neurological diseases. In this work, we employed the controlled cortical impact (CCI) model of experimental TBI in rat model to assess the temporal-global proteome changes after acute (1 day) and for the first time, subacute (7 days), post-injury time frame using the established cation-anion exchange chromatography-1D SDS gel electrophoresis LC-MS/MS platform for protein separation combined with discrete systems biology analyses to identify temporal biomarker changes related to this rat TBI model. Rather than focusing on any one individual molecular entity, we used in silico systems biology approach to understand the global dynamics that govern proteins that are differentially altered post-injury. In addition, gene ontology analysis of the proteomic data was conducted in order to categorize the proteins by molecular function, biological process, and cellular localization. Results show alterations in several proteins related to inflammatory responses and oxidative stress in both acute (1 day) and subacute (7 days) periods post-TBI. Moreover, results suggest a differential upregulation of neuroprotective proteins at 7 days post-CCI involved in cellular functions such as neurite growth, regeneration, and axonal guidance. Our study is among the first to assess temporal neuroproteome changes in the CCI model. Data presented here unveil potential neural biomarkers and therapeutic targets that could be used for diagnosis, for treatment and, most importantly, for temporal prognostic assessment following brain injury. Of interest, this work relies on in silico bioinformatics approach to draw its conclusion; further work is conducted for functional studies to validate and confirm the omics data obtained.
RESUMO
A major consequence of traumatic brain injury (TBI) is the rapid proteolytic degradation of structural cytoskeletal proteins. This process is largely reflected by the interruption of axonal transport as a result of extensive axonal injury leading to neuronal cell injury. Previous work from our group has described the extensive degradation of the axonally enriched cytoskeletal αII-spectrin protein which results in molecular signature breakdown products (BDPs) indicative of injury mechanisms and to specific protease activation both in vitro and in vivo. In the current study, we investigated the integrity of ßII-spectrin protein and its proteolytic profile both in primary rat cerebrocortical cell culture under apoptotic, necrotic, and excitotoxic challenge and extended to in vivo rat model of experimental TBI (controlled cortical impact model). Interestingly, our results revealed that the intact 260-kDa ßII-spectrin is degraded into major fragments (ßII-spectrin breakdown products (ßsBDPs)) of 110, 108, 85, and 80 kDa in rat brain (hippocampus and cortex) 48 h post-injury. These ßsBDP profiles were further characterized and compared to an in vitro ßII-spectrin fragmentation pattern of naive rat cortex lysate digested by calpain-2 and caspase-3. Results revealed that ßII-spectrin was degraded into major fragments of 110/85 kDa by calpain-2 activation and 108/80 kDa by caspase-3 activation. These data strongly support the hypothesis that in vivo activation of multiple protease system induces structural protein proteolysis involving ßII-spectrin proteolysis via a specific calpain and/or caspase-mediated pathway resulting in a signature, protease-specific ßsBDPs that are dependent upon the type of neural injury mechanism. This work extends on previous published work that discusses the interplay spectrin family (αII-spectrin and ßII-spectrin) and their susceptibility to protease proteolysis and their implication to neuronal cell death mechanisms.
Assuntos
Lesões Encefálicas/metabolismo , Calpaína/metabolismo , Caspase 3/metabolismo , Síndromes Neurotóxicas/metabolismo , Proteólise , Espectrina/metabolismo , Animais , Apoptose/efeitos dos fármacos , Lesões Encefálicas/patologia , Células Cultivadas , Córtex Cerebral/patologia , Hipocampo/patologia , Humanos , Immunoblotting , Masculino , Necrose , Síndromes Neurotóxicas/patologia , Neurotoxinas/toxicidade , Inibidores de Proteases/farmacologia , Proteólise/efeitos dos fármacos , Ratos Sprague-Dawley , Fatores de TempoRESUMO
Mass spectrometry (MS) has become the method of choice to study the proteome of brain injury. The high throughput nature of MS-based proteomic experiments generates massive amount of mass spectral data presenting great challenges in downstream interpretation. Currently, different bioinformatics platforms are available for functional analysis and data mining of MS-generated proteomic data. These tools provide a way to convert data sets to biologically interpretable results and functional outcomes. In this review, a brief overview of the currently available bioinformatics strategies applied to neuroproteomic studies is presented. Application of commercially available bioinformatics software to different brain injury studies demonstrates integration of the data mining and analysis applications into neuroproteomic workflows that can identify major protein markers as well as highlight the biological processes and molecular functions involved.
Assuntos
Lesões Encefálicas/metabolismo , Encéfalo/metabolismo , Biologia Computacional/métodos , Mapeamento de Peptídeos/métodos , Proteoma/metabolismo , Proteômica/métodos , Software , Algoritmos , Animais , HumanosRESUMO
The post-genomics era has brought about new Omics biotechnologies, such as proteomics and metabolomics, as well as their novel applications to personal genomics and the quantified self. These advances are now also catalyzing other and newer post-genomics innovations, leading to convergences between Omics and nanotechnology. In this work, we systematically contextualize and exemplify an emerging strand of post-genomics life sciences, namely, nanoproteomics and its applications in health and integrative biological systems. Nanotechnology has been utilized as a complementary component to revolutionize proteomics through different kinds of nanotechnology applications, including nanoporous structures, functionalized nanoparticles, quantum dots, and polymeric nanostructures. Those applications, though still in their infancy, have led to several highly sensitive diagnostics and new methods of drug delivery and targeted therapy for clinical use. The present article differs from previous analyses of nanoproteomics in that it offers an in-depth and comparative evaluation of the attendant biotechnology portfolio and their applications as seen through the lens of post-genomics life sciences and biomedicine. These include: (1) immunosensors for inflammatory, pathogenic, and autoimmune markers for infectious and autoimmune diseases, (2) amplified immunoassays for detection of cancer biomarkers, and (3) methods for targeted therapy and automatically adjusted drug delivery such as in experimental stroke and brain injury studies. As nanoproteomics becomes available both to the clinician at the bedside and the citizens who are increasingly interested in access to novel post-genomics diagnostics through initiatives such as the quantified self, we anticipate further breakthroughs in personalized and targeted medicine.
Assuntos
Nanotecnologia/métodos , Medicina de Precisão/métodos , Proteômica/métodos , Doenças Autoimunes/diagnóstico , Doenças Autoimunes/terapia , Disciplinas das Ciências Biológicas , Técnicas Biossensoriais , Doenças Transmissíveis/diagnóstico , Doenças Transmissíveis/terapia , Humanos , Imunoensaio , Terapia de Alvo Molecular , Nanoestruturas/uso terapêutico , Nanotecnologia/instrumentação , Nanotecnologia/tendências , Neoplasias/diagnóstico , Neoplasias/terapia , Medicina de Precisão/instrumentação , Proteômica/instrumentaçãoRESUMO
Intracerebral hemorrhage (ICH) is a devastating form of stroke leading to a high rate of death and disability worldwide. Although it has been hypothesized that much of the IHC insult occurs in the subacute period mediated via a series of complex pathophysiological cascades, the molecular mechanisms involved in ICH have not been systematically characterized. Among the best approaches to understand the underlying mechanisms of injury and recovery, protein dynamics assessment via proteomics/systems biology platforms represent one of the cardinal techniques optimized for mechanisms investigation and biomarker identification. A proteomics approach may provide a biomarker focused framework from which to identify candidate biomarkers of pathophysiological processes involved in brain injury after stroke. In this work, a neuroproteomic approach (LC-MS/MS) was applied to investigate altered expression of proteins that are induced in brain tissue 3 h after injury in a rat model of ICH. Data from sham and focal ischemic models were also obtained and used for comparison. Based on the differentially expressed protein profile, systems biology analysis was conducted to identify associated cellular processes and related interaction maps. After LC-MS/MS analysis of the 3 h brain lysates, 86 proteins were differentially expressed between hemorrhagic and sham tissues. Furthermore, 38 proteins were differentially expressed between ischemic and sham tissues. On the level of global pathway analysis, hemorrhagic stroke proteins were shown to be involved in autophagy, ischemia, necrosis, apoptosis, calpain activation, and cytokine secretion. Moreover, ischemic stroke proteins were related to cell death, ischemia, inflammation, oxidative stress, caspase activation and apoptotic injury. In conclusion, the proteomic responses identified in this study provide key information about target proteins involved in specific pathological pathways.
Assuntos
Encéfalo/metabolismo , Hemorragia Cerebral/metabolismo , Infarto da Artéria Cerebral Média/metabolismo , Proteômica/métodos , Acidente Vascular Cerebral/metabolismo , Biologia de Sistemas/métodos , Animais , Biomarcadores/metabolismo , Western Blotting , Encéfalo/patologia , Isquemia Encefálica/diagnóstico , Isquemia Encefálica/metabolismo , Isquemia Encefálica/patologia , Hemorragia Cerebral/diagnóstico , Hemorragia Cerebral/patologia , Modelos Animais de Doenças , Infarto da Artéria Cerebral Média/diagnóstico , Infarto da Artéria Cerebral Média/patologia , Masculino , Ratos , Ratos Sprague-Dawley , Índice de Gravidade de Doença , Transdução de Sinais , Acidente Vascular Cerebral/diagnóstico , Acidente Vascular Cerebral/patologiaRESUMO
Among the U.S. military personnel, blast injury is among the leading causes of brain injury. During the past decade, it has become apparent that even blast injury as a form of mild traumatic brain injury (mTBI) may lead to multiple different adverse outcomes, such as neuropsychiatric symptoms and long-term cognitive disability. Blast injury is characterized by blast overpressure, blast duration, and blast impulse. While the blast injuries of a victim close to the explosion will be severe, majority of victims are usually at a distance leading to milder form described as mild blast TBI (mbTBI). A major feature of mbTBI is its complex manifestation occurring in concert at different organ levels involving systemic, cerebral, neuronal, and neuropsychiatric responses; some of which are shared with other forms of brain trauma such as acute brain injury and other neuropsychiatric disorders such as post-traumatic stress disorder. The pathophysiology of blast injury exposure involves complex cascades of chronic psychological stress, autonomic dysfunction, and neuro/systemic inflammation. These factors render blast injury as an arduous challenge in terms of diagnosis and treatment as well as identification of sensitive and specific biomarkers distinguishing mTBI from other non-TBI pathologies and from neuropsychiatric disorders with similar symptoms. This is due to the "distinct" but shared and partially identified biochemical pathways and neuro-histopathological changes that might be linked to behavioral deficits observed. Taken together, this article aims to provide an overview of the current status of the cellular and pathological mechanisms involved in blast overpressure injury and argues for the urgent need to identify potential biomarkers that can hint at the different mechanisms involved.
RESUMO
The two primary categories of stroke, ischemic and hemorrhagic, both have fundamentally different mechanisms and thus different treatment options. These two stroke categories were applied to rat models to identify potential biomarkers that can distinguish between them. Ischemic stroke was induced by middle cerebral artery occlusion (MCAO) without reperfusion while hemorrhagic stroke was induced by injecting collagenase IV into the striatum. Brain hemispheres and biofluids were collected at two time points: 3 and 6h after stroke. Known molecules were tested on the rat samples via quantitative immunoblotting (injured brain, CSF) and Banyan's proprietary ELISA assays (CSF, serum). The injured brain quantitative analyses revealed that αII-spectrin breakdown products (SBDP150, SBDP145) were strongly increased after 6h ischemia. In CSF, SBDP145 and ubiquitin C-terminal hydrolase-L1 (UCH-L1) levels were elevated after 6h ischemic stroke detected by Western blot and ELISA. In serum UCH-L1 levels were increased after 3 and 6h of ischemia detected by ELISA. However, levels of those proteins in hemorrhagic stroke remain normal. In summary, in both the brain and the biofluids, SBDPs and UCH-L1 were elevated after ischemic but not hemorrhagic stroke. These molecules behaved differently in the two stroke models and thus may be capable of being differentiated.
Assuntos
Encéfalo/metabolismo , Espectrina/metabolismo , Acidente Vascular Cerebral/metabolismo , Ubiquitina Tiolesterase/metabolismo , Animais , Biomarcadores , Encéfalo/patologia , Modelos Animais de Doenças , Masculino , Ratos , Ratos Sprague-Dawley , Espectrina/líquido cefalorraquidiano , Acidente Vascular Cerebral/sangue , Acidente Vascular Cerebral/líquido cefalorraquidiano , Acidente Vascular Cerebral/diagnóstico , Acidente Vascular Cerebral/patologia , Ubiquitina Tiolesterase/sangue , Ubiquitina Tiolesterase/líquido cefalorraquidianoRESUMO
OBJECT: This study investigates a potential novel application of a selective cathepsin B and L inhibitor in experimental intracerebral hemorrhage (ICH) in rats. METHODS: Forty adult male Wistar rats received an ICH by stereotactic injection of 100 µl of autologous blood or sham via needle insertion into the right striatum. The rats were treated with a selective cathepsin B and L inhibitor (CP-1) or 1% dimethyl sulfoxide sterile saline intravenously at 2 and 4 hours after injury. Modified neurological severity scores were obtained and corner turn tests were performed at 1, 4, 7, and 14 days after ICH. The rats were sacrificed at 3 and 14 days after ICH for immunohistological analysis of tissue loss, neurogenesis, angiogenesis, and apoptosis. RESULTS: The animals treated with CP-1 demonstrated significantly reduced apoptosis as well as tissue loss compared with controls (p < 0.05 for each). Neurological function as assessed by modified neurological severity score and corner turn tests showed improvement after CP-1 treatment at 7 and 14 days (p < 0.05). Angiogenesis and neurogenesis parameters demonstrated improvement after CP-1 treatment compared with controls (p < 0.05) at 14 days. CONCLUSIONS: This study is the first report of treatment of ICH with a selective cathepsin B and L inhibitor. Cathepsin B and L inhibition has been shown to be beneficial after cerebral ischemia, likely because of its upstream regulation of the other prominent cysteine proteases, calpains, and caspases. While ICH may not induce a major component of ischemia, the cellular stress in the border zone may activate these proteolytic pathways. The observation that cathepsin B and L blockade is efficacious in this model is provocative for further investigation.
Assuntos
Catepsina B/antagonistas & inibidores , Catepsina L/antagonistas & inibidores , Hemorragia Cerebral/tratamento farmacológico , Diazometano/análogos & derivados , Dipeptídeos/uso terapêutico , Animais , Apoptose/efeitos dos fármacos , Comportamento Animal/efeitos dos fármacos , Comportamento Animal/fisiologia , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Hemorragia Cerebral/patologia , Hemorragia Cerebral/psicologia , Diazometano/uso terapêutico , Relação Dose-Resposta a Droga , Imuno-Histoquímica , Marcação In Situ das Extremidades Cortadas , Imageamento por Ressonância Magnética , Masculino , Neovascularização Fisiológica/fisiologia , Doenças do Sistema Nervoso/etiologia , Doenças do Sistema Nervoso/patologia , Ratos , Ratos Wistar , Sinaptofisina/metabolismo , Resultado do TratamentoRESUMO
BACKGROUND: Anti-angiogenic treatments of malignant tumors targeting vascular endothelial growth factor receptors (VEGFR) tyrosine kinase are being used in different early stages of clinical trials. Very recently, VEGFR tyrosine kinase inhibitor (Vetanalib, PTK787) was used in glioma patient in conjunction with chemotherapy and radiotherapy. However, changes in the tumor size, tumor vascular permeability, vascular density, expression of VEGFR2 and other angiogenic factors in response to PTK787 are not well documented. This study was to determine the changes in tumor size, vascular permeability, fractional plasma volume and expression of VEGFR2 in PTK787 treated U-251 glioma rat model by in vivo magnetic resonance imaging (MRI) and single photon emission computed tomography (SPECT). The findings were validated with histochemical and western blot studies. METHODOLOGIES AND PRINCIPAL FINDINGS: Seven days after implantation of U251 glioma cells, animals were treated with either PTK787 or vehicle-only for two weeks, and then tumor size, tumor vascular permeability transfer constant (K(trans)), fractional plasma volume (fPV) and expression of VEGFR2 and other relevant angiogenic factors were assessed by in vivo MRI and SPECT (Tc-99-HYNIC-VEGF), and by immunohistochemistry and western blot analysis. Dynamic contrast-enhanced MRI (DCE-MRI) using a high molecular weight contrast agent albumin-(GdDTPA) showed significantly increased K(trans) at the rim of the treated tumors compared to that of the central part of the treated as well as the untreated (vehicle treated) tumors. Size of the tumors was also increased in the treated group. Expression of VEGFR2 detected by Tc-99m-HYNIC-VEGF SPECT also showed significantly increased activity in the treated tumors. In PTK787-treated tumors, histological staining revealed increase in microvessel density in the close proximity to the tumor border. Western blot analysis indicated increased expression of VEGF, SDF-1, HIF-1alpha, VEGFR2, VEGFR3 and EGFR at the peripheral part of the treated tumors compared to that of central part of the treated tumors. Similar expression patters were not observed in vehicle treated tumors. CONCLUSION: These findings indicate that PTK787 treatment induced over expression of VEGF as well as the Flk-1/VEGFR2 receptor tyrosine kinase, especially at the rim of the tumor, as proven by DCE-MRI, SPECT imaging, immunohistochemistry and western blot.
Assuntos
Indutores da Angiogênese/metabolismo , Inibidores da Angiogênese/uso terapêutico , Neoplasias Encefálicas/tratamento farmacológico , Permeabilidade Capilar/efeitos dos fármacos , Glioma/tratamento farmacológico , Inibidores da Angiogênese/farmacologia , Animais , Western Blotting , Neoplasias Encefálicas/irrigação sanguínea , Neoplasias Encefálicas/patologia , Glioma/irrigação sanguínea , Glioma/patologia , Imuno-Histoquímica , Imageamento por Ressonância Magnética , Tomografia por Emissão de Pósitrons , RatosRESUMO
The serpin plasminogen activator inhibitor-1 (PAI-1) is a potential therapeutic target in cardiovascular and cancerous diseases. PAI-1 circulates in blood as a complex with vitronectin. A PAI-1 variant (N-((2-(iodoacetoxy)ethyl)-N-methyl)amino-7-nitrobenz-2-oxa-3-diazole (NBD) P9 PAI-1) with a fluorescent tag at the reactive center loop (RCL) was used to study the effects of vitronectin and monoclonal antibodies (mAbs) directed against alpha-helix F (Mab-2 and MA-55F4C12) on the reactions of PAI-1 with tissue-type and urokinase-type plasminogen activators. Both mAbs delay the RCL insertion and induce an increase in the stoichiometry of inhibition (SI) to 1.4-9.5. Binding of vitronectin to NBD P9 PAI-1 does not affect SI but results in a 2.0-6.5-fold decrease in the limiting rate constant (klim) of RCL insertion for urokinase-type plasminogen activator at pH 6.2-8.0 and for tissue-type plasminogen activator at pH 6.2. Binding of vitronectin to the complexes of NBD P9 PAI-1 with mAbs results in a decrease in klim and in a 1.5-22-fold increase in SI. Thus, vitronectin and mAbs demonstrated additivity in the effects on the reaction with target proteinases. The same step in the reaction mechanism remains limiting for the rate of RCL insertion in the absence and presence of Vn and mAbs. We hypothesize that vitronectin, bound to alpha-helix F on the side opposite to the epitopes of the mAbs, potentiates the mAb-induced delay in RCL insertion and the associated substrate behavior by selectively decreasing the rate constant for the inhibitory branch of PAI-1 reaction (ki). These results demonstrate that mAbs represent a valid approach for inactivation of vitronectin-bound PAI-1 in vivo.
Assuntos
Anticorpos Monoclonais/farmacologia , Peptídeo Hidrolases/metabolismo , Inibidor 1 de Ativador de Plasminogênio/química , Inibidor 1 de Ativador de Plasminogênio/metabolismo , Vitronectina/farmacologia , Anticorpos Monoclonais/imunologia , Humanos , Cinética , Ligantes , Modelos Moleculares , Inibidor 1 de Ativador de Plasminogênio/imunologia , Inibidores de Proteases/química , Inibidores de Proteases/imunologia , Inibidores de Proteases/metabolismo , Estrutura Secundária de Proteína , Especificidade por Substrato , Termodinâmica , Ativador de Plasminogênio Tecidual/metabolismo , Titulometria , Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Vitronectina/metabolismoRESUMO
Uncontrolled activation of calpain has been linked to tissue damage after neuronal and cardiac ischemias, traumatic spine and brain injuries, and multiple sclerosis and Alzheimer's disease. In vivo, the activity of calpain is regulated by its endogenous inhibitor calpastatin. The pathological role of calpain has been attributed to an imbalance between the activities of the protease and its inhibitor. Thus, it is possible that by reimposing functional control on the protease, the progression of calpain-mediated diseases could be slowed or eliminated. B27-WT is a 27-residue peptide (DPMSSTYIEELGKREVTIPPKYRELLA) derived from calpastatin that was previously shown to be a potent inhibitor of mu- and m-calpain. Recently, we identified two hot spots (Leu(11)-Gly(12) and Thr(17)-Ile(18)-Pro(19)) within which the amino acid residues that are key to B27-WT's bioactivity are clustered. In the work described here, the most critical residues of B27-WT, Leu(11) and Ile(18), were further probed to determine the nature of their interaction with calpain. Our results demonstrate that the side chains of both residues interact with hydrophobic pockets in calpain and that each of these interactions is indispensable for effective inhibition of calpain. Direct interactions involving the beta- and gamma-CH(2)- of the Leu(11) and Ile(18) side chains, respectively, rather than the degree of side chain branching or hydrophobicity, seemed to play a significant role in the peptide's ability to inhibit calpain. Furthermore, the minimum peptide sequence that still retained the calpain-inhibitory potency of B27-WT was found to be MSSTYIEELGKREVTIPPKYRELL.
Assuntos
Proteínas de Ligação ao Cálcio/química , Calpaína/antagonistas & inibidores , Inibidores de Cisteína Proteinase/química , Isoleucina/química , Leucina/química , Fragmentos de Peptídeos/química , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Autólise , Proteínas de Ligação ao Cálcio/síntese química , Inibidores de Cisteína Proteinase/síntese química , Humanos , Interações Hidrofóbicas e Hidrofílicas , Dados de Sequência Molecular , Fragmentos de Peptídeos/síntese química , Biblioteca de Peptídeos , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Relação Estrutura-Atividade , SuínosRESUMO
Calpastatin is the natural specific inhibitor of calpain. Recent research has linked uncontrolled calpain activation to tissue damage after neuronal and cardiac ischemias, traumatic spine and brain injuries, as well as Alzheimer's disease and cataract formation. An imbalance between the activities of calpain and calpastatin is believed to be responsible for the pathological role of calpain. An important key to understanding calpain regulation by calpastatin is to determine, at the molecular level, how calpastatin interacts with calpain to inhibit its enzymatic activity. A 27-residue peptide (DPMSSTYIEELGKREVTIPPKYRELLA) derived from subdomain 1B of the repetitive domains of calpain, named peptide B27-WT, was previously shown to be a potent inhibitor of mu- and m-calpain. In this report, a combination of beta-alanine scanning mutagenesis and kinetic measurements was used to probe, in a quantitative, systematic, and simultaneous fashion, the relative contribution of the amino acid side chain and backbone functionalities to the overall calpain-inhibitory activity of B27-WT. The study identified two "hot spots," Leu(11)-Gly(12) and Thr(17)-Ile(18)-Pro(19), in B27-WT within which the residues critical for inhibitory function are clustered. Mutation of any one of the key residues in either of the two hot spots resulted in a dramatic loss of inhibitory activity. Furthermore, it was shown that a restricted conformation of the Leu(11)-Gly(12) and Thr(17)-Ile(18)-Pro(19) backbones is required for the peptide inhibitory function. These results suggest a plausible model in which the two hot spots are situated at or near the interface(s) of the calpain-calpastatin complex and act in a concerted fashion to inhibit calpain. The information on the specific contribution of the amide bond and side chain of each key residue to the bioactivity of B27-WT will contribute to a better understanding of the mechanism of calpain inhibition and lead to novel and effective therapies based on the specific inhibition of dysregulated or overactivated calpain.