Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 20
Filtrar
1.
Acta Neuropathol ; 147(1): 32, 2024 02 06.
Artigo em Inglês | MEDLINE | ID: mdl-38319380

RESUMO

Synapse loss correlates with cognitive decline in Alzheimer's disease, and soluble oligomeric amyloid beta (Aß) is implicated in synaptic dysfunction and loss. An important knowledge gap is the lack of understanding of how Aß leads to synapse degeneration. In particular, there has been difficulty in determining whether there is a synaptic receptor that binds Aß and mediates toxicity. While many candidates have been observed in model systems, their relevance to human AD brain remains unknown. This is in part due to methodological limitations preventing visualization of Aß binding at individual synapses. To overcome this limitation, we combined two high resolution microscopy techniques: array tomography and Förster resonance energy transfer (FRET) to image over 1 million individual synaptic terminals in temporal cortex from AD (n = 11) and control cases (n = 9). Within presynapses and post-synaptic densities, oligomeric Aß generates a FRET signal with transmembrane protein 97. Further, Aß generates a FRET signal with cellular prion protein, and post-synaptic density 95 within post synapses. Transmembrane protein 97 is also present in a higher proportion of post synapses in Alzheimer's brain compared to controls. We inhibited Aß/transmembrane protein 97 interaction in a mouse model of amyloidopathy by treating with the allosteric modulator CT1812. CT1812 drug concentration correlated negatively with synaptic FRET signal between transmembrane protein 97 and Aß. In human-induced pluripotent stem cell derived neurons, transmembrane protein 97 is present in synapses and colocalizes with Aß when neurons are challenged with human Alzheimer's brain homogenate. Transcriptional changes are induced by Aß including changes in genes involved in neurodegeneration and neuroinflammation. CT1812 treatment of these neurons caused changes in gene sets involved in synaptic function. These data support a role for transmembrane protein 97 in the synaptic binding of Aß in human Alzheimer's disease brain where it may mediate synaptotoxicity.


Assuntos
Doença de Alzheimer , Disfunção Cognitiva , Proteínas de Membrana , Animais , Humanos , Camundongos , Peptídeos beta-Amiloides , Encéfalo , Sinapses , Proteínas de Membrana/metabolismo
3.
Int J Mol Sci ; 23(15)2022 Jul 27.
Artigo em Inglês | MEDLINE | ID: mdl-35897835

RESUMO

A series of σ2R compounds containing benzimidazolone and diazacycloalkane cores was synthesized and evaluated in radioligand binding assays. Replacing the piperazine moiety in a lead compound with diazaspiroalkanes and the fused octahydropyrrolo[3,4-b] pyrrole ring system resulted in a loss in affinity for the σ2R. On the other hand, the bridged 2,5-diazabicyclo[2.2.1]heptane, 1,4-diazepine, and a 3-aminoazetidine analog possessed nanomolar affinities for the σ2R. Computational chemistry studies were also conducted with the recently published crystal structure of the σ2R/TMEM97 and revealed that hydrogen bond interactions with ASP29 and π-stacking interactions with TYR150 were largely responsible for the high binding affinity of small molecules to this protein.


Assuntos
Receptores sigma , Ligantes , Piperazina , Ensaio Radioligante , Receptores sigma/metabolismo , Relação Estrutura-Atividade
4.
J Med Chem ; 65(8): 6261-6272, 2022 04 28.
Artigo em Inglês | MEDLINE | ID: mdl-35404616

RESUMO

In this study, a panel of 46 compounds containing five different scaffolds known to have high σ2 receptor affinity were screened. 6,7-Dimethoxy-2-[4-(4-methoxyphenyl)butan-2-yl]-1,2,3,4-tetrahydroisoquinoline [(±)-7] (Ki for σ1 = 48.4 ± 7.7 nM, and Ki for σ2 = 0.59 ± 0.02 nM) and its desmethyl analogue, (±)-8 (Ki for σ1 = 108 ± 35 nM, and Ki for σ2 = 4.92 ± 0.59 nM), showed excellent binding affinity and subtype selectivity for σ2 receptors. In vitro cell binding indicated that σ2 receptor binding of [11C]-(±)-7 and [11C]-(±)-8 was dependent on TMEM97 protein expression. In PET studies, the peak brain uptake of [11C]-(±)-7 (8.28 ± 2.52%ID/cc) was higher than that of [11C]-(±)-8 (4.25 ± 0.97%ID/cc) with specific distribution in the cortex and hypothalamus. Brain uptake or tissue binding was selectively inhibited by ligands with different σ2 receptor binding affinities. The results suggest [11C]-(±)-7 can be used as a PET radiotracer for imaging the function of σ2 receptors in central nervous system disorders.


Assuntos
Receptores sigma , Tetra-Hidroisoquinolinas , Encéfalo/diagnóstico por imagem , Encéfalo/metabolismo , Ligantes , Tomografia por Emissão de Pósitrons , Compostos Radiofarmacêuticos/química , Tetra-Hidroisoquinolinas/química
5.
ACS Med Chem Lett ; 12(9): 1389-1395, 2021 Sep 09.
Artigo em Inglês | MEDLINE | ID: mdl-34531947

RESUMO

An unbiased phenotypic neuronal assay was developed to measure the synaptotoxic effects of soluble Aß oligomers. A collection of CNS druglike small molecules prepared by conditioned extraction was screened. Compounds that prevented and reversed synaptotoxic effects of Aß oligomers in neurons were discovered to bind to the sigma-2 receptor complex. Select development compounds displaced receptor-bound Aß oligomers, rescued synapses, and restored cognitive function in transgenic hAPP Swe/Ldn mice. Our first-in-class orally administered small molecule investigational drug 7 (CT1812) has been advanced to Phase II clinical studies for Alzheimer's disease.

6.
Alzheimers Dement ; 17(8): 1365-1382, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-33559354

RESUMO

INTRODUCTION: Amyloid beta (Aß) oligomers are one of the most toxic structural forms of the Aß protein and are hypothesized to cause synaptotoxicity and memory failure as they build up in Alzheimer's disease (AD) patients' brain tissue. We previously demonstrated that antagonists of the sigma-2 receptor complex effectively block Aß oligomer toxicity. CT1812 is an orally bioavailable, brain penetrant small molecule antagonist of the sigma-2 receptor complex that appears safe and well tolerated in healthy elderly volunteers. We tested CT1812's effect on Aß oligomer pathobiology in preclinical AD models and evaluated CT1812's impact on cerebrospinal fluid (CSF) protein biomarkers in mild to moderate AD patients in a clinical trial (ClinicalTrials.gov NCT02907567). METHODS: Experiments were performed to measure the impact of CT1812 versus vehicle on Aß oligomer binding to synapses in vitro, to human AD patient post mortem brain tissue ex vivo, and in living APPSwe /PS1dE9 transgenic mice in vivo. Additional experiments were performed to measure the impact of CT1812 versus vehicle on Aß oligomer-induced deficits in membrane trafficking rate, synapse number, and protein expression in mature hippocampal/cortical neurons in vitro. The impact of CT1812 on cognitive function was measured in transgenic Thy1 huAPPSwe/Lnd+ and wild-type littermates. A multicenter, double-blind, placebo-controlled parallel group trial was performed to evaluate the safety, tolerability, and impact on protein biomarker expression of CT1812 or placebo given once daily for 28 days to AD patients (Mini-Mental State Examination 18-26). CSF protein expression was measured by liquid chromatography with tandem mass spectrometry or enzyme-linked immunosorbent assay in samples drawn prior to dosing (Day 0) and at end of dosing (Day 28) and compared within each patient and between pooled treated versus placebo-treated dosing groups. RESULTS: CT1812 significantly and dose-dependently displaced Aß oligomers bound to synaptic receptors in three independent preclinical models of AD, facilitated oligomer clearance into the CSF, increased synaptic number and protein expression in neurons, and improved cognitive performance in transgenic mice. CT1812 significantly increased CSF concentrations of Aß oligomers in AD patient CSF, reduced concentrations of synaptic proteins and phosphorylated tau fragments, and reversed expression of many AD-related proteins dysregulated in CSF. DISCUSSION: These preclinical studies demonstrate the novel disease-modifying mechanism of action of CT1812 against AD and Aß oligomers. The clinical results are consistent with preclinical data and provide evidence of target engagement and impact on fundamental disease-related signaling pathways in AD patients, supporting further development of CT1812.


Assuntos
Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/líquido cefalorraquidiano , Biomarcadores/líquido cefalorraquidiano , Cognição/efeitos dos fármacos , Camundongos Transgênicos , Receptores sigma/antagonistas & inibidores , Idoso , Animais , Encéfalo/metabolismo , Método Duplo-Cego , Ensaio de Imunoadsorção Enzimática , Hipocampo/metabolismo , Humanos , Masculino , Camundongos , Neurônios/metabolismo , Sinapses/metabolismo
7.
J Neurosci Res ; 99(4): 1161-1176, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33480104

RESUMO

α-Synuclein oligomers are thought to have a pivotal role in sporadic and familial Parkinson's disease (PD) and related α-synucleinopathies, causing dysregulation of protein trafficking, autophagy/lysosomal function, and protein clearance, as well as synaptic function impairment underlying motor and cognitive symptoms of PD. Moreover, trans-synaptic spread of α-synuclein oligomers is hypothesized to mediate disease progression. Therapeutic approaches that effectively block α-synuclein oligomer-induced pathogenesis are urgently needed. Here, we show for the first time that α-synuclein species isolated from human PD patient brain and recombinant α-synuclein oligomers caused similar deficits in lipid vesicle trafficking rates in cultured rat neurons and glia, while α-synuclein species isolated from non-PD human control brain samples did not. Recombinant α-synuclein oligomers also increased neuronal expression of lysosomal-associated membrane protein-2A (LAMP-2A), the lysosomal receptor that has a critical role in chaperone-mediated autophagy. Unbiased screening of several small molecule libraries (including the NIH Clinical Collection) identified sigma-2 receptor antagonists as the most effective at blocking α-synuclein oligomer-induced trafficking deficits and LAMP-2A upregulation in a dose-dependent manner. These results indicate that antagonists of the sigma-2 receptor complex may alleviate α-synuclein oligomer-induced neurotoxicity and are a novel therapeutic approach for disease modification in PD and related α-synucleinopathies.


Assuntos
Doença de Parkinson/metabolismo , Receptores sigma/antagonistas & inibidores , Receptores sigma/metabolismo , alfa-Sinucleína/metabolismo , Idoso , Idoso de 80 Anos ou mais , Animais , Autofagia/efeitos dos fármacos , Encéfalo/metabolismo , Feminino , Ensaios de Triagem em Larga Escala , Humanos , Metabolismo dos Lipídeos , Proteína 2 de Membrana Associada ao Lisossomo/metabolismo , Masculino , Doença de Parkinson/patologia , Cultura Primária de Células , Ratos , Ratos Sprague-Dawley , Proteínas de Transporte Vesicular/metabolismo , alfa-Sinucleína/farmacologia
8.
J Neurochem ; 157(4): 1316-1330, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33025581

RESUMO

Several mutations conferring protection against Alzheimer's disease (AD) have been described, none as profound as the A673T mutation, where carriers are four times less likely to get AD compared to noncarriers. This mutation results in reduced amyloid beta (Aß) protein production in vitro and lower lifetime Aß concentration in carriers. Better understanding of the protective mechanisms of the mutation may provide important insights into AD pathophysiology and identify productive therapeutic intervention strategies for disease modification. Aß(1-42) protein forms oligomers that bind saturably to a single receptor site on neuronal synapses, initiating the downstream toxicities observed in AD. Decreased formation, toxicity, or stability of soluble Aß oligomers, or reduction of synaptic binding of these oligomers, may combine with overall lower Aß concentration to underlie A673T's disease protecting mechanism. To investigate these possibilities, we compared the formation rate of soluble oligomers made from Icelandic A673T mutant and wild type (wt) Aß(1-42) synthetic protein, the amount and intensity of oligomer bound to mature primary rat hippocampal/cortical neuronal synapses, and the potency of bound oligomers to impact trafficking rate in neurons in vitro using a physiologically relevant oligomer preparation method. At equal protein concentrations, mutant protein forms approximately 50% or fewer oligomers of high molecular weight (>50 kDa) compared to wt protein. Mutant oligomers are twice as potent at altering the cellular vesicle trafficking rate as wt at equivalent concentrations, however, mutant oligomers have a >4-fold lower binding affinity to synaptic receptors (Kd  = 1,950 vs. 442 nM). The net effect of these differences is a lower overall toxicity at a given concentration. This study demonstrates for the first time that mutant A673T Aß oligomers prepared with this method have fundamentally different assembly characteristics and biological impact from wt protein and indicates that its disease protecting mechanism may result primarily from the mutant protein's much lower binding affinity to synaptic receptors. This suggests that therapeutics that effectively reduce oligomer binding to synapses in the brain may be beneficial in AD.


Assuntos
Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/genética , Peptídeos beta-Amiloides/metabolismo , Neurônios/metabolismo , Animais , Humanos , Ligação Proteica , Transporte Proteico/fisiologia , Ratos , Ratos Sprague-Dawley
9.
eNeuro ; 7(6)2020.
Artigo em Inglês | MEDLINE | ID: mdl-33028631

RESUMO

The σ-2 receptor (S2R) complex has been implicated in CNS disorders ranging from anxiety and depression to neurodegenerative disorders such as Alzheimer's disease (AD). The proteins comprising the S2R complex impact processes including autophagy, cholesterol synthesis, progesterone signaling, lipid membrane-bound protein trafficking, and receptor stabilization at the cell surface. While there has been much progress in understanding the role of S2R in cellular processes and its potential therapeutic value, a great deal remains unknown. The International Symposium on Sigma-2 Receptors is held in conjunction with the annual Society for Neuroscience (SfN) conference to promote collaboration and advance the field of S2R research. This review summarizes updates presented at the Fourth International Symposium on Sigma-2 Receptors: Role in Health and Disease, a Satellite Symposium held at the 2019 SfN conference. Interdisciplinary members of the S2R research community presented both previously published and preliminary results from ongoing studies of the role of S2R in cellular metabolism, the anatomic and cellular expression patterns of S2R, the relationship between S2R and amyloid ß (Aß) in AD, the role of S2R complex protein PGRMC1 in health and disease, and the efforts to design new S2R ligands for the purposes of research and drug development. The proceedings from this symposium are reported here as an update on the field of S2R research, as well as to highlight the value of the symposia that occur yearly in conjunction with the SfN conference.


Assuntos
Doença de Alzheimer , Receptores sigma , Peptídeos beta-Amiloides , Humanos , Proteínas de Membrana , Progesterona , Receptores de Progesterona
10.
Alzheimers Res Ther ; 12(1): 21, 2020 03 02.
Artigo em Inglês | MEDLINE | ID: mdl-32122400

RESUMO

BACKGROUND: Synapse damage and loss are fundamental to the pathophysiology of Alzheimer's disease (AD) and lead to reduced cognitive function. The goal of this review is to address the challenges of forging new clinical development approaches for AD therapeutics that can demonstrate reduction of synapse damage or loss. The key points of this review include the following: Synapse loss is a downstream effect of amyloidosis, tauopathy, inflammation, and other mechanisms occurring in AD.Synapse loss correlates most strongly with cognitive decline in AD because synaptic function underlies cognitive performance.Compounds that halt or reduce synapse damage or loss have a strong rationale as treatments of AD.Biomarkers that measure synapse degeneration or loss in patients will facilitate clinical development of such drugs.The ability of methods to sensitively measure synapse density in the brain of a living patient through synaptic vesicle glycoprotein 2A (SV2A) positron emission tomography (PET) imaging, concentrations of synaptic proteins (e.g., neurogranin or synaptotagmin) in the cerebrospinal fluid (CSF), or functional imaging techniques such as quantitative electroencephalography (qEEG) provides a compelling case to use these types of measurements as biomarkers that quantify synapse damage or loss in clinical trials in AD. CONCLUSION: A number of emerging biomarkers are able to measure synapse injury and loss in the brain and may correlate with cognitive function in AD. These biomarkers hold promise both for use in diagnostics and in the measurement of therapeutic successes.


Assuntos
Doença de Alzheimer/líquido cefalorraquidiano , Doença de Alzheimer/diagnóstico por imagem , Doença de Alzheimer/patologia , Biomarcadores/líquido cefalorraquidiano , Sinapses/patologia , Eletroencefalografia/métodos , Neuroimagem Funcional/métodos , Humanos , Tomografia por Emissão de Pósitrons/métodos
11.
Alzheimers Dement (N Y) ; 5: 20-26, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30723776

RESUMO

BACKGROUND: Elayta (CT1812) is a novel allosteric antagonist of the sigma-2 receptor complex that prevents and displaces binding of Aß oligomers to neurons. By stopping a key initiating event in Alzheimer's disease, this first-in-class drug candidate mitigates downstream synaptotoxicity and restores cognitive function in aged transgenic mouse models of Alzheimer's disease. METHODS: A phase 1, two-part single and multiple ascending dose study was conducted in 7 and 4 cohorts of healthy human subjects, respectively. In part A, healthy, young subjects (<65 years old) received CT1812 doses ranging from 10 to 1120 mg (6:2 active to placebo [A:P] per cohort). In part B, subjects were administered 280, 560, and 840 mg once daily for 14 days (8:2 A:P per cohort). An elderly cohort, aged 65-75 years, was dosed at 560 mg once daily for 14 days (7:2 A:P). Serum concentrations of CT1812 in part B were measured on day 3 and 14 and cerebrospinal fluid concentrations on day 7 or 9. Cognitive testing was performed in the healthy elderly cohort at baseline and at day 14 of treatment. RESULTS: Treatment with CT1812 was well tolerated in all cohorts. Adverse events were mild to moderate in severity and included headache and GI tract symptoms. Plasma concentrations of drug were dose proportional across two orders of magnitude with minimal accumulation over 14 days. Cognitive scores in the healthy elderly cohort were similar before and after treatment. CONCLUSIONS: CT1812 was well tolerated with single dose administration up to 1120 mg and with multiple dose administration up to 840 mg and 560 mg in healthy young and healthy elderly subjects, respectively. CT1812 is currently being studied in early phase 2 trials in patients with Alzheimer's disease.

12.
PLoS One ; 9(11): e111898, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25390368

RESUMO

Synaptic dysfunction and loss caused by age-dependent accumulation of synaptotoxic beta amyloid (Abeta) 1-42 oligomers is proposed to underlie cognitive decline in Alzheimer's disease (AD). Alterations in membrane trafficking induced by Abeta oligomers mediates reduction in neuronal surface receptor expression that is the basis for inhibition of electrophysiological measures of synaptic plasticity and thus learning and memory. We have utilized phenotypic screens in mature, in vitro cultures of rat brain cells to identify small molecules which block or prevent the binding and effects of Abeta oligomers. Synthetic Abeta oligomers bind saturably to a single site on neuronal synapses and induce deficits in membrane trafficking in neuronal cultures with an EC50 that corresponds to its binding affinity. The therapeutic lead compounds we have found are pharmacological antagonists of Abeta oligomers, reducing the binding of Abeta oligomers to neurons in vitro, preventing spine loss in neurons and preventing and treating oligomer-induced deficits in membrane trafficking. These molecules are highly brain penetrant and prevent and restore cognitive deficits in mouse models of Alzheimer's disease. Counter-screening these compounds against a broad panel of potential CNS targets revealed they are highly potent and specific ligands of the sigma-2/PGRMC1 receptor. Brain concentrations of the compounds corresponding to greater than 80% receptor occupancy at the sigma-2/PGRMC1 receptor restore cognitive function in transgenic hAPP Swe/Ldn mice. These studies demonstrate that synthetic and human-derived Abeta oligomers act as pharmacologically-behaved ligands at neuronal receptors--i.e. they exhibit saturable binding to a target, they exert a functional effect related to their binding and their displacement by small molecule antagonists blocks their functional effect. The first-in-class small molecule receptor antagonists described here restore memory to normal in multiple AD models and sustain improvement long-term, representing a novel mechanism of action for disease-modifying Alzheimer's therapeutics.


Assuntos
Doença de Alzheimer/tratamento farmacológico , Peptídeos beta-Amiloides/química , Neurônios/metabolismo , Fragmentos de Peptídeos/química , Sinapses/efeitos dos fármacos , Doença de Alzheimer/metabolismo , Animais , Encéfalo/efeitos dos fármacos , Química Farmacêutica , Cognição/efeitos dos fármacos , Transtornos Cognitivos/tratamento farmacológico , Desenho de Fármacos , Ensaio de Imunoadsorção Enzimática , Humanos , Camundongos , Camundongos Transgênicos , Neuroglia/metabolismo , Ligação Proteica , Transporte Proteico , Ratos , Ratos Sprague-Dawley , Sinapses/metabolismo
13.
PLoS One ; 9(11): e111899, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25390692

RESUMO

Amyloid beta (Abeta) 1-42 oligomers accumulate in brains of patients with Mild Cognitive Impairment (MCI) and disrupt synaptic plasticity processes that underlie memory formation. Synaptic binding of Abeta oligomers to several putative receptor proteins is reported to inhibit long-term potentiation, affect membrane trafficking and induce reversible spine loss in neurons, leading to impaired cognitive performance and ultimately to anterograde amnesia in the early stages of Alzheimer's disease (AD). We have identified a receptor not previously associated with AD that mediates the binding of Abeta oligomers to neurons, and describe novel therapeutic antagonists of this receptor capable of blocking Abeta toxic effects on synapses in vitro and cognitive deficits in vivo. Knockdown of sigma-2/PGRMC1 (progesterone receptor membrane component 1) protein expression in vitro using siRNA results in a highly correlated reduction in binding of exogenous Abeta oligomers to neurons of more than 90%. Expression of sigma-2/PGRMC1 is upregulated in vitro by treatment with Abeta oligomers, and is dysregulated in Alzheimer's disease patients' brain compared to age-matched, normal individuals. Specific, high affinity small molecule receptor antagonists and antibodies raised against specific regions on this receptor can displace synthetic Abeta oligomer binding to synaptic puncta in vitro and displace endogenous human AD patient oligomers from brain tissue sections in a dose-dependent manner. These receptor antagonists prevent and reverse the effects of Abeta oligomers on membrane trafficking and synapse loss in vitro and cognitive deficits in AD mouse models. These findings suggest sigma-2/PGRMC1 receptors mediate saturable oligomer binding to synaptic puncta on neurons and that brain penetrant, small molecules can displace endogenous and synthetic oligomers and improve cognitive deficits in AD models. We propose that sigma-2/PGRMC1 is a key mediator of the pathological effects of Abeta oligomers in AD and is a tractable target for small molecule disease-modifying therapeutics.


Assuntos
Doença de Alzheimer/tratamento farmacológico , Peptídeos beta-Amiloides/química , Proteínas de Membrana/metabolismo , Fragmentos de Peptídeos/química , Receptores de Progesterona/metabolismo , Sinapses/efeitos dos fármacos , Doença de Alzheimer/metabolismo , Animais , Autorradiografia , Encéfalo/metabolismo , Membrana Celular/metabolismo , Cognição/efeitos dos fármacos , Transtornos Cognitivos/tratamento farmacológico , Humanos , Proteínas de Membrana/genética , Camundongos , Neurônios/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , RNA Interferente Pequeno/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores de Progesterona/genética , Sinapses/metabolismo
14.
J Orthop Res ; 27(6): 793-9, 2009 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-19051248

RESUMO

Articular cartilage is an avascular tissue with chondrocytes in the deeper zones existing under conditions of sustained hypoxia. Using a hypoxic chamber to provide controlled hypoxia, this study was performed to determine whether sustained hypoxia enhances the production of cartilage matrix proteins. Freshly isolated primary bovine articular chondrocytes were encapsulated in three-dimensional alginate beads and maintained at 2% oxygen with media changes using media pre-equilibrated to 2% oxygen. Immunolocalization of HIF-1alpha was performed to verify hypoxic conditions. Sustained hypoxia resulted in an increase in proteoglycan synthesis after only 1 day, as measured by 35S-sulfate incorporation. This increase was maintained for the duration of the 17 day study. After 17 days of hypoxic culture, increases in total type II collagen and COL2A1 gene expression were probed by indirect immunofluorescence, type II collagen ELISA, and real-time qPCR; in addition, increased glycosaminoglycan deposition was observed as determined by chemical analysis. These studies show that sustained hypoxia enhances articular chondrocyte matrix synthesis and viability in three-dimensional alginate culture.


Assuntos
Condrócitos/citologia , Condrócitos/metabolismo , Proteínas da Matriz Extracelular/genética , Matriz Extracelular/metabolismo , Oxigênio/farmacologia , Alginatos , Animais , Cartilagem Articular/citologia , Bovinos , Técnicas de Cultura de Células/métodos , Hipóxia Celular/fisiologia , Sobrevivência Celular/fisiologia , Células Cultivadas , Colágeno Tipo II/genética , Colágeno Tipo II/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Expressão Gênica/fisiologia , Ácido Glucurônico , Ácidos Hexurônicos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Microesferas , Reação em Cadeia da Polimerase , Proteoglicanas/genética , Proteoglicanas/metabolismo
15.
J Orthop Res ; 26(5): 643-50, 2008 May.
Artigo em Inglês | MEDLINE | ID: mdl-18050310

RESUMO

Fresh osteochondral allografts are an important treatment option for the repair of full-thickness articular cartilage defects. Viable chondrocytes within the transplanted tissue are considered important to maintaining matrix integrity. The purpose of this study is to determine whether an increase in pH decreases chondrocyte viability during cold storage and whether equilibration of Dulbecco's modified Eagle's medium (DMEM) in 5% CO(2) normalizes pH and increases chondrocyte survival during storage at 4 degrees C. Freshly isolated bovine articular chondrocytes cultured in alginate beads were stored for up to 5 days at 4 degrees C or 37 degrees C in DMEM exposed to ambient air or in DMEM equilibrated with 5% CO(2). Chondrocyte viability was determined by flow cytometry. Physiologic pH was maintained when DMEM was equilibrated with 5% CO(2), while pH increased in ambient air. After 5 days of storage at 4 degrees C, chondrocyte necrosis was higher when stored in ambient air than if equilibrated with 5% CO(2). No decrease in chondrocyte viability was observed with storage at 37 degrees C. In addition, chondrocyte viability in bovine cartilage osteochondral cores was examined after storage for 14 days at 4 degrees C in DMEM with and without HEPES, and with and without 5% CO(2). Under these conditions, the superficial layer of chondrocytes was more viable when stored in DMEM with HEPES or DMEM equilibrated with 5% CO(2) than when stored in DMEM in ambient air. This data shows that an increase in pH decreased bovine chondrocyte viability when refrigerated at 4 degrees C in DMEM, and that optimization of CO(2) normalized pH and improved chondrocyte viability during cold storage in DMEM.


Assuntos
Dióxido de Carbono , Condrócitos/citologia , Temperatura Baixa , Preservação de Tecido/métodos , Animais , Bovinos , Sobrevivência Celular , Meios de Cultura , Concentração de Íons de Hidrogênio
16.
J Biomed Opt ; 12(5): 051703, 2007.
Artigo em Inglês | MEDLINE | ID: mdl-17994876

RESUMO

A series of bench to operating room studies was conducted to determine whether it is feasible to use optical coherence tomography (OCT) clinically to diagnose potentially reversible early cartilage degeneration. A human cadaver study was performed to confirm the reproducibility of OCT imaging and grading based on identification of changes to cartilage OCT form birefringence using a polarized OCT system approved for clinical use. Segregation of grossly normal appearing human articular cartilage into two groups based on the presence or absence of OCT form birefringence showed that cartilage without OCT form birefringence had reduced ability to increase proteoglycan synthetic activity in response to the anabolic growth factor IGF-1. The bench data further show that IGF-1 insensitivity in cartilage without OCT form birefringence was reversible. To show clinical feasibility, OCT was then used arthroscopically in 19 human subjects. Clinical results confirmed that differences to OCT form birefringence observed in ex vivo study were detectable during arthroscopic surgery. More prevalent loss of cartilage OCT form birefringence was observed in cartilage of human subjects in groups more likely to have cartilage degeneration. This series of integrated bench to bedside studies demonstrates translational feasibility to use OCT for clinical studies on whether human cartilage degeneration can be diagnosed early enough for intervention that may delay or prevent the onset of osteoarthritis.


Assuntos
Artroscopia/métodos , Cartilagem Articular/patologia , Cartilagem Articular/cirurgia , Osteoartrite/patologia , Osteoartrite/cirurgia , Tomografia de Coerência Óptica/métodos , Humanos , Seleção de Pacientes , Cuidados Pré-Operatórios/métodos , Reprodutibilidade dos Testes , Sensibilidade e Especificidade
17.
Arthroscopy ; 22(7): 693-9, 2006 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-16843803

RESUMO

PURPOSE: Intra-articular use of 0.5% bupivacaine is common in arthroscopic surgery. This study was conducted to test the hypotheses that (1) 0.5% bupivacaine is toxic to articular chondrocytes, and (2) the intact articular surface protects chondrocytes from the effects of short-term exposure to 0.5% bupivacaine. METHODS: Freshly isolated bovine articular chondrocytes were prepared into alginate bead cultures and were treated with 0.5% bupivacaine solution or 0.9% saline for 15, 30 or 60 minutes, washed, and returned to growth media. Chondrocytes were recovered from alginate 1 hour, 1 day, and 1 week after bupivacaine exposure; they were fluorescently labeled to identify apoptotic and dead cells and were analyzed by flow cytometry. Twelve osteochondral cores were harvested from bovine knees. The superficial 1 mm of cartilage was removed from 6 cores (top-off). Intact and top-off cores were submerged in 0.9% saline or 0.5% bupivacaine solution for 30 minutes and then maintained in chondrocyte growth media for 24 hours. Live-cell/dead-cell fluorescent imaging was assessed using confocal microscopy. RESULTS: Greater than 99% chondrocyte death/apoptosis was observed in all bupivacaine-exposed alginate bead cultures compared with 20% cell death in saline-treated controls (P < .05). Osteochondral cores with intact surfaces treated with 0.5% bupivacaine showed 42% dead chondrocytes. When the articular surface was removed, 0.5% bupivacaine resulted in increased cell death, with 75% dead chondrocytes (P < .05). CONCLUSIONS: Results show that 0.5% bupivacaine solution is cytotoxic to bovine articular chondrocytes and articular cartilage in vitro after only 15 to 30 minutes' exposure. The intact bovine articular surface has some chondroprotective effects. CLINICAL RELEVANCE: Because healthy chondrocytes are important for maintenance of the cartilage matrix, chondrocyte loss may contribute to cartilage degeneration. This study shows a cytotoxic effect of 0.5% bupivacaine solution on bovine articular chondrocytes in vitro. Although these results cannot be directly extrapolated to the clinical setting, the data suggest that caution should be exercised in the intra-articular use of 0.5% bupivacaine.


Assuntos
Anestésicos Locais/administração & dosagem , Anestésicos Locais/toxicidade , Bupivacaína/administração & dosagem , Bupivacaína/toxicidade , Cartilagem Articular/efeitos dos fármacos , Condrócitos/efeitos dos fármacos , Alginatos , Animais , Cartilagem Articular/citologia , Cartilagem Articular/fisiologia , Bovinos , Sobrevivência Celular/efeitos dos fármacos , Condrócitos/patologia , Relação Dose-Resposta a Droga , Citometria de Fluxo , Ácido Glucurônico , Ácidos Hexurônicos , Técnicas Histológicas , Técnicas In Vitro , Microscopia Confocal , Microesferas , Fatores de Tempo
18.
Arthritis Rheum ; 52(10): 3181-91, 2005 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-16200622

RESUMO

OBJECTIVE: To determine whether oxygen-dependent activation patterns of hypoxia-inducible factor 1alpha (HIF-1alpha) observed in vascularized tissues are conserved within avascular and hypoxic articular cartilage and whether HIF-1alpha affects cartilage matrix synthesis. METHODS: Explants of bovine articular cartilage and primary chondrocytes were exposed to normoxia (21% O2), hypoxia (2% O2), and simulated hypoxia (21% O2 plus CoCl2). Western blot and immunofluorescence analyses of HIF-1alpha were performed to determine HIF-1alpha activation patterns. To simulate cartilage loss from disease or injury, the top layers of cartilage were removed from osteochondral explants, and the residual cartilage was assessed for HIF-1alpha immunolocalization and proteoglycan synthesis. RESULTS: We demonstrated continuous nuclear translocation of HIF-1alpha in deeper layers of intact articular cartilage. HIF-1alpha was not completely degraded in chondrocytes exposed to normoxia, but rather, colocalized to the Golgi complex, a finding not previously reported for any cell type. Following alteration of the oxygen gradient by removal of the top layers of cartilage, predominantly perinuclear HIF-1alpha was found in the deeper layers. Restoration of intranuclear HIF-1alpha to these areas was achieved by hypoxia and simulated hypoxia. Under conditions in which HIF-1alpha was inactivated, matrix synthetic activity was altered (P < 0.0001) compared with control cartilage. CONCLUSION: These findings demonstrate that hypoxia-dependent activation of HIF-1alpha is highly conserved and that changes in oxygen tensions following cartilage loss from injury or disease alter cartilage metabolism in part by changing HIF-1alpha activity. The discovery of tonic activation of HIF-1alpha within intact articular cartilage underscores its potential importance to cartilage homeostasis.


Assuntos
Cartilagem Articular/metabolismo , Condrócitos/metabolismo , Homeostase/fisiologia , Fatores de Transcrição/metabolismo , Animais , Cartilagem Articular/irrigação sanguínea , Cartilagem Articular/citologia , Bovinos , Núcleo Celular/metabolismo , Sobrevivência Celular/fisiologia , Células Cultivadas , Condrócitos/citologia , Complexo de Golgi/metabolismo , Hipóxia/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia , Osteoartrite/metabolismo , Oxigênio/metabolismo , Proteoglicanas/biossíntese
19.
Exp Hematol ; 33(8): 865-72, 2005 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-16038778

RESUMO

OBJECTIVE: The objective of the present study was to investigate the potential of application of growth factor genes to induce chondrogenic differentiation of human-derived mesenchymal stem cells (MSCs). The growth factor genes evaluated in the present study were transforming growth factor 1 (TGF-beta1) and insulin-like growth factor 1 (IGF-1). METHODS: Human MSCs were transduced with the adenoviral vectors carrying either TGF-beta1 or IGF-1 (AdTGF-beta1 and AdIGF-1 respectively) or a combination of both growth factor genes at different multiplicities of infection (MOI) and were then made into pellets. Pellets were also made from nontransduced cells and maintained in culture medium supplemented with 10 ng/mL of TGF-beta1. At specified time points, histological analysis, cartilage matrix gene expression, and immunofluorescence were performed to determine the extent of chondrogenic differentiation. RESULTS: MSCs transduced with the AdTGF-beta1 demonstrated robust chondrogenic differentiation, while those made from AdIGF-1 did not. AdTGF-beta1 pellets demonstrated aggrecan gene expression as early as day 3 of pellet culture, while type II collagen gene expression was detected by day 10 of culture. The AdIGF-1, alone or in combination with TGF-beta1 pellets, did not show any type II collagen gene expression at any time point. By immunofluoresecence, type X collagen was distributed throughout the matrix in TGF-beta1 protein pellets while the growth factor gene pellets displayed scant staining. CONCLUSION: The results suggest that sustained administration of TGF-beta1 may be more effective in suppressing terminal differentiation than intermittent dosing and thus effective for cartilage repair.


Assuntos
Diferenciação Celular/genética , Condrogênese/genética , Regulação da Expressão Gênica , Fator de Crescimento Insulin-Like I/genética , Células-Tronco Mesenquimais , Fator de Crescimento Transformador beta/genética , Adenoviridae , Células Cultivadas , Colágeno Tipo II/biossíntese , Colágeno Tipo II/genética , Colágeno Tipo X/biossíntese , Colágeno Tipo X/genética , Técnicas de Transferência de Genes , Humanos , Fator de Crescimento Insulin-Like I/metabolismo , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/fisiologia , Fator de Crescimento Transformador beta/metabolismo , Fator de Crescimento Transformador beta1
20.
Anal Biochem ; 316(2): 181-91, 2003 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-12711339

RESUMO

The application of microchip capillary electrophoresis (CE) to the assay of extracellular signal-regulated protein kinase (ERK) is presented. In this assay, ERK catalyzes the transfer of gamma-phosphate from adenosine 5(')-triphosphate to the threonine residue of a fluorescently labeled nonapeptide (APRTPGGRR), and the phosphorylated and nonphosphorylated peptides were detected by fluorescence. The phosphorylated and nonphosphorylated peptides and the internal standard were separated within 20s, and the increase in magnitude of the phosphorylated peptide peak was monitored to assess ERK activity. ERK reactions were prepared off-chip and analyzed on a single-lane glass microchip fabricated by standard methods. It was demonstrated that microchip CE could be used to measure endogenous amounts of ERK by spiking known concentrations of recombinant ERK2 into the lysates of serum-starved human umbilical vein endothelial cells (HUVEC) and recovering between 90 and 100% for all samples. Endogenous ERK activity was determined by microchip where HUVEC were stimulated with 500pM vascular endothelial growth factor (VEGF) at different times before cell lysis. The results showed a transient VEGF-mediated ERK activation that peaked at 10min, which was consistent with previous reports using conventional techniques. The microchip assay provided a rapid, accurate, and precise alternative to conventional methods of determining endogenous ERK activity.


Assuntos
Eletroforese Capilar/métodos , Endotélio Vascular/enzimologia , Proteínas Quinases Ativadas por Mitógeno/análise , Células Cultivadas , Eletroforese Capilar/instrumentação , Eletroforese Capilar/normas , Endotélio Vascular/efeitos dos fármacos , Fluorescência , Humanos , Cinética , Microquímica , Proteína Quinase 1 Ativada por Mitógeno/análise , Proteína Quinase 1 Ativada por Mitógeno/química , Proteína Quinase 1 Ativada por Mitógeno/normas , Proteínas Quinases Ativadas por Mitógeno/química , Proteínas Quinases Ativadas por Mitógeno/normas , Fosforilação , Padrões de Referência , Reprodutibilidade dos Testes , Fatores de Tempo , Veias Umbilicais , Fator A de Crescimento do Endotélio Vascular/farmacologia
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA