RESUMO
INTRODUCTION/AIMS: The precise relationship between molecular mimicry and tissue-specific autoimmunity is unknown. Major histocompatibility complex (MHC) class II antigen presenting cell-CD4+ T-cell receptor complex interactions are necessary for adaptive immunity. This study aimed to determine the role of endoneurial endothelial cell MHC class II in autoimmune polyneuropathy. METHODS: Cryopreserved Guillain-Barré syndrome (GBS) patient sural nerve biopsies and sciatic nerves from the severe murine experimental autoimmune neuritis (sm-EAN) GBS model were studied. Cultured conditional ready MHC Class II antigen A-alpha chain (H2-Aa) embryonic stem cells were used to generate H2-Aaflox/+ C57BL/6 mice. Mice were backcrossed and intercrossed to the SJL background to generate H2-Aaflox/flox SJL mice, bred with hemizygous Tamoxifen-inducible von Willebrand factor Cre recombinase (vWF-iCre/+) SJL mice to generate H2-Aaflox/flox; vWF-iCre/+ mice to study microvascular endothelial cell adaptive immune responses. Sm-EAN was induced in Tamoxifen-treated H2-Aaflox/flox; vWF-iCre/+, H2-Aaflox/flox; +/+, H2-Aa+/+; vWF-iCre/+ and untreated H2-Aaflox/flox; vWF-iCre/+ adult female SJL mice. Neurobehavioral, electrophysiological and histopathological assessments were performed at predefined time points. RESULTS: Endoneurial endothelial cell MHC class II expression was observed in normal and inflamed human and mouse peripheral nerves. Tamoxifen-treated H2-Aaflox/flox; vWF-iCre/+ mice were resistant to sm-EAN despite extensive MHC class II expression in lymphoid and non-lymphoid tissues. DISCUSSION: A conditional MHC class II knockout mouse to study cell- and time-dependent adaptive immune responses in vivo was developed. Initial studies show microvascular endothelial cell MHC class II expression is necessary for peripheral nerve specific autoimmunity, as advocated by human in vitro adaptive immunity and ex vivo transplant rejection studies.
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Imunidade Adaptativa , Antígenos de Histocompatibilidade Classe II , Camundongos Endogâmicos C57BL , Camundongos Knockout , Animais , Camundongos , Antígenos de Histocompatibilidade Classe II/metabolismo , Antígenos de Histocompatibilidade Classe II/genética , Humanos , Síndrome de Guillain-Barré/imunologia , Síndrome de Guillain-Barré/genética , Síndrome de Guillain-Barré/patologia , Neurite Autoimune Experimental/imunologia , Neurite Autoimune Experimental/patologia , Neurite Autoimune Experimental/genética , Nervo Isquiático/imunologia , Nervo Isquiático/metabolismo , Nervo Isquiático/patologia , Nervos Periféricos/imunologia , Nervos Periféricos/metabolismo , Nervos Periféricos/patologia , Feminino , Células Endoteliais/imunologia , Células Endoteliais/metabolismo , Nervo Sural/patologia , Nervo Sural/imunologia , Masculino , Fatores de TempoRESUMO
Endosomal sorting plays a fundamental role in directing neural development. By altering the temporal and spatial distribution of membrane receptors, endosomes regulate signaling pathways that control the differentiation and function of neural cells. Several genes linked to inherited demyelinating peripheral neuropathies, known as Charcot-Marie-Tooth (CMT) disease, encode proteins that directly interact with components of the endosomal sorting complex required for transport (ESCRT). Our previous studies demonstrated that a point mutation in the ESCRT component hepatocyte growth-factor-regulated tyrosine kinase substrate (HGS), an endosomal scaffolding protein that identifies internalized cargo to be sorted by the endosome, causes a peripheral neuropathy in the neurodevelopmentally impaired teetering mice. Here, we constructed a Schwann cell-specific deletion of Hgs to determine the role of endosomal sorting during myelination. Inactivation of HGS in Schwann cells resulted in motor and sensory deficits, slowed nerve conduction velocities, delayed myelination and hypomyelinated axons, all of which occur in demyelinating forms of CMT. Consistent with a delay in Schwann cell maturation, HGS-deficient sciatic nerves displayed increased mRNA levels for several promyelinating genes and decreased mRNA levels for genes that serve as markers of myelinating Schwann cells. Loss of HGS also altered the abundance and activation of the ERBB2/3 receptors, which are essential for Schwann cell development. We therefore hypothesize that HGS plays a critical role in endosomal sorting of the ERBB2/3 receptors during Schwann cell maturation, which further implicates endosomal dysfunction in inherited peripheral neuropathies.SIGNIFICANCE STATEMENT Schwann cells myelinate peripheral axons, and defects in Schwann cell function cause inherited demyelinating peripheral neuropathies known as CMT. Although many CMT-linked mutations are in genes that encode putative endosomal proteins, little is known about the requirements of endosomal sorting during myelination. In this study, we demonstrate that loss of HGS disrupts the endosomal sorting pathway in Schwann cells, resulting in hypomyelination, aberrant myelin sheaths, and impairment of the ERBB2/3 receptor pathway. These findings suggest that defective endosomal trafficking of internalized cell surface receptors may be a common mechanism contributing to demyelinating CMT.
Assuntos
Doença de Charcot-Marie-Tooth , Animais , Doença de Charcot-Marie-Tooth/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte , Endossomos/metabolismo , Camundongos , Doenças do Sistema Nervoso Periférico , RNA Mensageiro , Células de Schwann/metabolismoRESUMO
Originally identified in fibroblasts, Protease Inhibitor (PI)16 was recently shown to be crucial for the development of neuropathic pain via effects on blood-nerve barrier permeability and leukocyte infiltration, though its impact on inflammatory pain has not been established. Using the complete Freund's Adjuvant inflammatory pain model, we show that Pi16-/- mice are protected against sustained inflammatory pain. Accordingly, intrathecal delivery of a PI16 neutralizing antibody in wild-type mice prevented sustained CFA pain. In contrast to neuropathic pain models, we did not observe any changes in blood-nerve barrier permeability due to PI16 deletion. Instead, Pi16-/- mice display reduced macrophage density in the CFA-injected hindpaw. Furthermore, there was a significant bias toward CD206hi (anti-inflammatory) macrophages in the hindpaw and associated dorsal root ganglia. Following CFA, intrathecal depletion of CD206+ macrophages using mannosylated clodronate liposomes promoted sustained pain in Pi16-/- mice. Similarly, an IL-10 neutralizing antibody also promoted sustained CFA pain in the Pi16-/ when administered intrathecally. Collectively, our results point to fibroblast-derived PI16 mediating substantial differences in macrophage phenotype in the pain neuroaxis under conditions of inflammation. The co-expression of PI16 alongside fibroblast markers in human DRG raise the likelihood that a similar mechanism operates in human inflammatory pain states. Collectively, our findings may have implications for targeting fibroblast-immune cell crosstalk for the treatment of chronic pain.
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Dor Crônica , Neuralgia , Camundongos , Humanos , Animais , Inflamação , Macrófagos , Fibroblastos , Anticorpos Neutralizantes/farmacologia , Gânglios Espinais , Hiperalgesia , Proteínas de Transporte , GlicoproteínasRESUMO
INTRODUCTION: Our aim in this study was to identify the prevalence and clinical characteristics of LRP4/agrin-antibody-positive double-seronegative myasthenia gravis (DNMG). METHODS: DNMG patients at 16 sites in the United States were tested for LRP4 and agrin antibodies, and the clinical data were collected. RESULTS: Of 181 DNMG patients, 27 (14.9%) were positive for either low-density lipoprotein receptor-related protein 4 (LRP4) or agrin antibodies. Twenty-three DNMG patients (12.7%) were positive for both antibodies. More antibody-positive patients presented with generalized symptoms (69%) compared with antibody-negative patients (43%) (P ≤ .02). Antibody-positive patients' maximum classification on the Myasthenia Gravis Foundation of America (MGFA) scale was significantly higher than that for antibody-negative patients (P ≤ .005). Seventy percent of antibody-positive patients were classified as MGFA class III, IV, or V compared with 39% of antibody-negative patients. Most LRP4- and agrin-antibody-positive patients (24 of 27, 89%) developed generalized myathenia gravis (MG), but with standard MG treatment 81.5% (22 of 27) improved to MGFA class I or II during a mean follow-up of 11 years. DISCUSSION: Antibody-positive patients had more severe clinical disease than antibody-negative patients. Most DNMG patients responded to standard therapy regardless of antibody status.
Assuntos
Agrina/imunologia , Autoanticorpos , Proteínas Relacionadas a Receptor de LDL/imunologia , Miastenia Gravis/diagnóstico , Adulto , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Miastenia Gravis/epidemiologia , Miastenia Gravis/imunologia , Prevalência , Avaliação de Sintomas , Estados UnidosRESUMO
The blood-nerve barrier (BNB) formed by tight junction-forming endoneurial microvessels located in the innermost compartment of peripheral nerves, and the perineurium serve to maintain the internal microenvironment required for normal signal transduction. The specific molecular components that define the normal adult human BNB are not fully known. Guided by data derived from the adult human BNB transcriptome, we evaluated the in situ expression of 25 junctional complex, transporter, cell membrane, and cytoskeletal proteins in four histologically normal adult sural nerves by indirect fluorescent immunohistochemistry to determine proteins specifically expressed by restrictive endoneurial microvascular endothelium. Using Ulex Europaeus Agglutinin-1 expression to detect endothelial cells, we ascertained that the selected proteins were uniformly expressed in ≥90% of endoneurial microvessels. P-glycoprotein (also known as adenosine triphosphate-binding cassette subfamily B member 1) and solute carrier family 1 member 1 demonstrated restricted expression by endoneurial endothelium only, with classic tight junction protein claudin-5 also expressed on fenestrated epineurial macrovessels, and vascular-specific adherens junction protein cadherin-5 also expressed by the perineurium. The expression profiles of the selected proteins provide significant insight into the molecular composition of normal adult peripheral nerves. Further work is required to elucidate the human adult BNB molecular signature in order to better understand its development and devise strategies to restore function in peripheral neuropathies.
Assuntos
Barreira Hematoneural/metabolismo , Microvasos/metabolismo , Nervos Periféricos/metabolismo , Transcriptoma , Idoso , Aglutininas/metabolismo , Células Endoteliais/metabolismo , Feminino , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-IdadeRESUMO
Microvascular barrier permeability to water is an essential biophysical property required for the homeostatic maintenance of unique tissue microenvironments. This is of particular importance in peripheral nerves where strict control of ionic concentrations is needed for axonal signal transduction. Previous studies have associated inflammation, trauma, toxin exposure and metabolic disease with increases in water influx and hydrostatic pressure in peripheral nerves with resultant endoneurial edema that may impair axonal function. The regulation of water permeability across endoneurial microvessels that form the blood-nerve barrier (BNB) is poorly understood. Variations exist in apparatus and methods used to measure hydraulic conductivity. The objective of the study was to develop a simplified hydraulic conductivity system using commercially available components to evaluate the BNB. We determined the mean hydraulic conductivity of cultured confluent primary and immortalized human endoneurial endothelial cell layers as 2.00×10-7 and 2.17×10-7cm/s/cm H2O respectively, consistent with restrictive microvascular endothelial cells in vitro. We also determined the mean hydraulic conductivity of immortalized human brain microvascular endothelial cell layers, a commonly used blood-brain barrier (BBB) cell line, as 0.20×10-7cm/s/cm H2O, implying a mean 10-fold higher resistance to transendothelial water flux in the brain compared to peripheral nerves. To our knowledge, this is the first reported measurement of human BNB and BBB hydraulic conductivities. This model represents an important tool to further characterize the human BNB and deduce the molecular determinants and signaling mechanisms responsible for BNB hydraulic conductivity in normal and disease states in vitro.
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Barreira Hematoneural , Permeabilidade Capilar , Técnicas Citológicas , Animais , Antígenos Transformantes de Poliomavirus/imunologia , Axônios/metabolismo , Bovinos , Linhagem Celular , Permeabilidade da Membrana Celular , Citocinas/metabolismo , Células Endoteliais/citologia , Fibroblastos/metabolismo , Homeostase , Humanos , Inflamação , Camundongos , Nervos Periféricos , Permeabilidade , Ratos , Ovinos , Transdução de Sinais , Suínos , Junções Íntimas/metabolismo , Água/químicaRESUMO
Peripheral neuroinflammation is characterized by hematogenous mononuclear leukocyte infiltration into peripheral nerves. Despite significant clinical knowledge, advancements in molecular biology and progress in developing specific drugs for inflammatory disorders such as rheumatoid arthritis, inflammatory bowel disease, and multiple sclerosis, there are currently no specific therapies that modulate pathogenic peripheral nerve inflammation. Modeling leukocyte trafficking at the blood-nerve barrier using a reliable human in vitro model and potential intravital microscopy techniques in representative animal models guided by human observational data should facilitate the targeted modulation of the complex inflammatory cascade needed to develop safe and efficacious therapeutics for immune-mediated neuropathies and chronic neuropathic pain.
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Barreira Hematoencefálica/fisiopatologia , Terapia Genética/métodos , Leucócitos/fisiologia , Neurite (Inflamação)/terapia , Sistema Nervoso Periférico/patologia , Animais , Humanos , Neurite (Inflamação)/genética , Sistema Nervoso Periférico/metabolismoRESUMO
The molecular determinants and mechanisms involved in leukocyte trafficking across the blood-nerve barrier (BNB) in the acute inflammatory demyelinating polyradiculoneuropathy (AIDP) variant of Guillain-Barré syndrome are incompletely understood. Prior work using a flow-dependent in vitro human BNB model demonstrated a crucial role for αM-integrin (CD11b)-intercellular adhesion molecule-1 interactions in AIDP patient leukocyte trafficking. The aim of this study is to directly investigate the biological relevance of CD11b in AIDP pathogenesis. Immunohistochemistry was performed on three AIDP patient sural nerve biopsies to evaluate endoneurial leukocyte CD11b expression. A severe murine experimental autoimmune neuritis (sm-EAN) model was utilized to determine the functional role of CD11b in leukocyte trafficking in vivo and determine its effect on neurobehavioral measures of disease severity, electrophysiological assessments of axonal integrity and myelination and histopathological measures of peripheral nerve inflammatory demyelination. Time-lapse video microscopy and electron microscopy were employed to observe structural alterations at the BNB during AIDP patient leukocyte trafficking in vitro and in situ, respectively. Large clusters of endoneurial CD11b+ leukocytes associated with demyelinating axons were observed in AIDP patient sural nerves. Leukocyte CD11b expression was upregulated during sm-EAN. 5 mg/kg of a function-neutralizing monoclonal rat anti-mouse CD11b antibody administered after sm-EAN disease onset significantly ameliorated disease severity, as well as electrophysiological and histopathological parameters of inflammatory demyelination compared to vehicle- and isotype antibody-treated mice. Consistent with in vitro observations of leukocyte trafficking at the BNB, electron micrographs of AIDP patient sural nerves demonstrated intact electron-dense endoneurial microvascular intercellular junctions during paracellular mononuclear leukocyte transmigration. Our data support a crucial pathogenic role of CD11b in AIDP leukocyte trafficking, providing a potential therapeutic target for demyelinating variants of Guillain-Barré syndrome.
Assuntos
Antígeno CD11b/genética , Síndrome de Guillain-Barré/metabolismo , Síndrome de Guillain-Barré/patologia , Leucócitos/metabolismo , Animais , Anticorpos/farmacologia , Antígenos CD/metabolismo , Antígenos de Diferenciação/metabolismo , Antígeno CD11b/imunologia , Antígeno CD11b/metabolismo , Rastreamento de Células , Modelos Animais de Doenças , Estimulação Elétrica , Feminino , Citometria de Fluxo , Regulação da Expressão Gênica/efeitos dos fármacos , Síndrome de Guillain-Barré/tratamento farmacológico , Síndrome de Guillain-Barré/genética , Humanos , Imunoglobulinas Intravenosas/uso terapêutico , Leucócitos/patologia , Leucócitos/ultraestrutura , Masculino , Camundongos , Microscopia Eletrônica , Condução Nervosa , Proteínas de Neurofilamentos/metabolismo , Subunidade beta da Proteína Ligante de Cálcio S100/metabolismo , Nervo Isquiático/metabolismo , Nervo Isquiático/fisiopatologia , Estatísticas não ParamétricasRESUMO
Inflammatory neuropathies encompass groups of heterogeneous disorders characterized by pathogenic immune-mediated hematogenous leukocyte infiltration of peripheral nerves, nerve roots or both, with resultant demyelination or axonal degeneration or both. Inflammatory neuropathies may be divided into three major disease categories: Guillain-Barré syndrome (particularly the acute inflammatory demyelinating polyradiculoneuropathy variant), chronic inflammatory demyelinating polyradiculoneuropathy and nonsystemic vasculitic neuropathy (or peripheral nerve vasculitis). Despite major advances in molecular biology, pathology and genetics, the pathogenesis of these disorders remains elusive. There is insufficient knowledge on the mechanisms of hematogenous leukocyte trafficking into the peripheral nervous system to guide the development of specific molecular therapies for immune-mediated inflammatory neuropathies compared to disorders such as psoriasis, inflammatory bowel disease, rheumatoid arthritis or multiple sclerosis. The recent isolation and characterization of human endoneurial endothelial cells that form the blood-nerve barrier provides an opportunity to elucidate leukocyte-endothelial cell interactions critical to the pathogenesis of inflammatory neuropathies at the interface between the systemic circulation and peripheral nerve endoneurium. This review discusses our current knowledge of the classic pathological features of inflammatory neuropathies, attempts at molecular classification and genetic determinants, the utilization of in vitro and in vivo animal models to determine pathogenic mechanisms at the interface between the systemic circulation and the peripheral nervous system relevant to these disorders and prospects for future potential molecular pathology biomarkers and targets for specific therapeutic intervention.
Assuntos
Doenças Autoimunes do Sistema Nervoso/metabolismo , Doenças Autoimunes do Sistema Nervoso/terapia , Animais , Doenças Autoimunes do Sistema Nervoso/genética , Doenças Autoimunes do Sistema Nervoso/patologia , Biomarcadores/metabolismo , Humanos , Leucócitos/fisiologiaRESUMO
The spontaneous autoimmune peripheral polyneuropathy (SAPP) model in B7-2 knockout nonobese diabetic mice mimics a progressive and unremitting course of chronic inflammatory demyelinating polyradiculoneuropathy (CIDP). In this study, bone marrow-derived dendritic cells (DCs) were transduced to express vasoactive intestinal polypeptide (VIP) using a lentiviral vector (LV-VIP). These transduced DCs (LV-VIP-DCs) were then injected intravenously (i.v.) into 16-week-old (before disease onset) and 21-week-old (after disease onset) SAPP mice in order to prevent or attenuate the disease. Outcome measures included behavioral tests, clinical and histological scoring, electrophysiology, real-time PCR, flow cytometry analyses, and enzyme-linked immunosorbent assay. LV-VIP-DCs were recruited to the inflamed sciatic nerve and reduced the expression of inflammatory cytokines. A single injection of LV-VIP-DC delayed the onset of disease, stabilized, and attenuated clinical signs correlating with ameliorated behavioral functions, reduced nerve demyelination, and improved nerve conduction. This proof-of-principle study is an important step potentially leading to a clinical translational study using DCs expressing VIP in cases of CIDP refractory to standard immunosuppressive therapy.
Assuntos
Células Dendríticas/metabolismo , Células Dendríticas/fisiologia , Doenças do Sistema Nervoso Periférico/terapia , Polineuropatias/terapia , Peptídeo Intestinal Vasoativo/metabolismo , Animais , Células Cultivadas , Células Dendríticas/citologia , Masculino , CamundongosRESUMO
BACKGROUND: Heterotopic ossification (HO) is the process of bone formation at a nonskeletal site. Recently, we showed that the earliest steps occur in sensory nerves. We now extend these studies by identifying unique osteogenic progenitors within the endoneurial compartment of sensory nerves. QUESTIONS/PURPOSES: We asked: (1) What is the nature of the osteoprogenitor in the endoneurium of peripheral nerves? (2) How do osteoprogenitors travel from the nerve to the site of new bone formation? METHODS: HO was induced by intramuscular injection of Ad5BMP-2-transduced cells in mice. Osteoprogenitors were identified through immunohistochemistry and then quantified and further characterized by fluorescence-activated cell sorting and immunocytochemistry. The kinetics of the appearance of markers of extravasation was determined by quantitative reverse transcription-polymerase chain reaction. In each experiment mice were injected with bone morphogenetic protein-2 (BMP-2)-producing cells (experimental) or with cells transduced with empty vector or, in some cases, a group receiving no injection (control). RESULTS: Induction of HO leads to the expression, within 24 hours, of osteoblast-specific transcription factors in cells in the endoneurium followed by their coordinate disappearance from the nerve at 48 hours. They reappear in blood also at 48 hours after induction. During vessel entrance they begin to express the tight junction molecule, claudin 5. The cells expressing both the osteoblast-specific transcription factor, osterix, as well as claudin 5, then disappear from circulation at approximately 3 to 4 days by extravasation into the site of new bone formation. These endoneurial osteoprogenitors express neural markers PDGFRα, musashi-1, and the low-affinity nerve growth factor receptor p75(NTR) as well as the endothelial marker Tie-2. In a key experiment, cells that were obtained from mice that were injected with cells transduced with an empty vector, at 2 days after injection, contained 0.83% (SD, 0.07; 95% confidence interval [CI], 0.59-1.05) cells expressing claudin 5. However, cells that were obtained from mice 2 days after injection of BMP-2-producing cells contained 4.5% cells expressing claudin 5 (SD, 0.72%; 95% CI, 2.01-6.94; p < 0.0015). Further analysis revealed that all of the cells expressing claudin 5 were found to be positive for osteoblast-specific markers, whereas cells not expressing claudin 5 were negative for these same markers. CONCLUSIONS: The findings suggest that the endoneurial progenitors are the major osteogenic precursors that are used for HO. They exit the nerve through the endoneurial vessels, flow through vessels to the site of new bone formation, and then extravasate out of the vessels into this site. CLINICAL RELEVANCE: The biogenesis of osteoblasts in HO is very different than expected and shows that HO is, at least in part, a neurological disorder. This could result in a major shift in orthopaedic methodologies to prevent or treat this disease. The fact that nerves are intimately involved in the process may also provide clues that will lead to an explanation of the clinical fact that HO often occurs as a result of traumatic brain injury.
Assuntos
Linhagem da Célula , Células-Tronco Neurais/patologia , Ossificação Heterotópica/patologia , Osteoblastos/patologia , Células Receptoras Sensoriais/patologia , Adenoviridae/genética , Animais , Biomarcadores/metabolismo , Proteína Morfogenética Óssea 2/biossíntese , Proteína Morfogenética Óssea 2/genética , Movimento Celular , Modelos Animais de Doenças , Regulação da Expressão Gênica , Vetores Genéticos , Cinética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Células-Tronco Neurais/metabolismo , Ossificação Heterotópica/genética , Ossificação Heterotópica/metabolismo , Osteoblastos/metabolismo , RNA Mensageiro/metabolismo , Células Receptoras Sensoriais/metabolismo , Transdução de Sinais , Transdução GenéticaRESUMO
Immune-mediated demyelinating polyneuropathies (IMDPs) are rare disorders in which dysregulated adaptive immune responses cause peripheral nerve demyelinating inflammation and axonal injury in susceptible individuals. Despite significant advances in understanding IMDP pathogenesis guided by patient data and representative mammalian models, specific therapies are lacking. Significant knowledge gaps in IMDP pathogenesis still exist, e.g. precise antigen(s) and mechanisms that initially trigger immune system activation and identification of large population disease susceptibility factors. The initial directional cues for antigen-specific effector or autoreactive leukocyte trafficking into peripheral nerves are also unknown. An overview of current animal models, with emphasis on the experimental autoimmune neuritis and spontaneous autoimmune peripheral polyneuropathy models, is provided. Insights on the initial directional cues for peripheral nerve tissue specific autoimmunity using a novel Major Histocompatibility Complex class II conditional knockout mouse strain are also discussed, suggesting an essential research tool to study cell- and time-dependent adaptive immunity in autoimmune diseases.
Assuntos
Modelos Animais de Doenças , Animais , Humanos , Camundongos , Neurite Autoimune Experimental/imunologia , Camundongos Knockout , Autoimunidade , Polineuropatias/imunologia , Polineuropatias/etiologia , Imunidade Adaptativa , Antígenos de Histocompatibilidade Classe II/imunologia , Antígenos de Histocompatibilidade Classe II/metabolismoRESUMO
Mouse models are invaluable to understanding fundamental mechanisms in vascular biology during development, in health and different disease states. Several constitutive or inducible models that selectively knockout or knock in genes in vascular endothelial cells exist; however, functional and phenotypic differences exist between microvascular and macrovascular endothelial cells in different organs. In order to study microvascular endothelial cell-specific biological processes, we developed a Tamoxifen-inducible von Willebrand Factor (vWF) Cre recombinase mouse in the SJL background. The transgene consists of the human vWF promoter with the microvascular endothelial cell-selective 734 base pair sequence to drive Cre recombinase fused to a mutant estrogen ligand-binding domain [ERT2] that requires Tamoxifen for activity (CreERT2) followed by a polyadenylation (polyA) signal. We initially observed Tamoxifen-inducible restricted bone marrow megakaryocyte and sciatic nerve microvascular endothelial cell Cre recombinase expression in offspring of a mixed strain hemizygous C57BL/6-SJL founder mouse bred with mT/mG mice, with >90% bone marrow megakaryocyte expression efficiency. Founder mouse offspring were backcrossed to the SJL background by speed congenics, and intercrossed for >10 generations to develop hemizygous Tamoxifen-inducible vWF Cre recombinase (vWF-iCre/+) SJL mice with stable transgene insertion in chromosome 1. Microvascular endothelial cell-specific Cre recombinase expression occurred in the sciatic nerves, brains, spleens, kidneys and gastrocnemius muscles of adult vWF-iCre/+ SJL mice bred with Ai14 mice, with retained low level bone marrow and splenic megakaryocyte expression. This novel mouse strain would support hypothesis-driven mechanistic studies to decipher the role(s) of specific genes transcribed by microvascular endothelial cells during development, as well as in physiologic and pathophysiologic states in an organ- and time-dependent manner.
Assuntos
Células Endoteliais , Integrases , Tamoxifeno , Fator de von Willebrand , Animais , Feminino , Humanos , Masculino , Camundongos , Células Endoteliais/metabolismo , Integrases/genética , Integrases/metabolismo , Megacariócitos/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microvasos/metabolismo , Regiões Promotoras Genéticas , Tamoxifeno/farmacologia , Fator de von Willebrand/metabolismo , Fator de von Willebrand/genéticaRESUMO
Diabetic peripheral neuropathy (DPN) is a prevalent complication of diabetes mellitus that is caused by metabolic toxicity to peripheral axons. We aimed to gain deep mechanistic insight into the disease process using bulk and spatial RNA sequencing on tibial and sural nerves recovered from lower leg amputations in a mostly diabetic population. First, our approach comparing mixed sensory and motor tibial and purely sensory sural nerves shows key pathway differences in affected nerves, with distinct immunological features observed in sural nerves. Second, spatial transcriptomics analysis of sural nerves reveals substantial shifts in endothelial and immune cell types associated with severe axonal loss. We also find clear evidence of neuronal gene transcript changes, like PRPH, in nerves with axonal loss suggesting perturbed RNA transport into distal sensory axons. This motivated further investigation into neuronal mRNA localization in peripheral nerve axons generating clear evidence of robust localization of mRNAs such as SCN9A and TRPV1 in human sensory axons. Our work gives new insight into the altered cellular and transcriptomic profiles in human nerves in DPN and highlights the importance of sensory axon mRNA transport as an unappreciated potential contributor to peripheral nerve degeneration.
RESUMO
The internal microenvironment in peripheral nerves is highly regulated in order to maintain normal axonal impulse transmission to or from the central nervous system. In humans, this regulation is facilitated by specialized tight junction (TJ)-forming endoneurial microvascular endothelial cells and perineurial myofibroblasts that form multiple concentric layers around nerve fascicles. The endoneurial endothelial cells come in direct contact with circulating blood and, thus, can be considered the blood-nerve barrier (BNB). Studies on the molecular and biophysical properties of the human BNB in vivo or in situ are limited. Owing to the recent isolation of primary human endoneurial endothelial cells and the development of simian virus 40 large T-antigen immortalized cell lines, data are emerging on the structural and functional characteristics of these cells. These data aim to increase our understanding of how solutes, macromolecules, nutrients and hematogenous leukocytes gain access into or are restricted from the endoneurium of peripheral nerves. These concepts have clinical relevance in understanding normal peripheral nerve homeostasis, the response of peripheral nerves to external insult and stresses such as drugs and toxins and the pathogenesis of peripheral neuropathies. This review discusses current knowledge in this nascent and exciting field of microvascular biology.
Assuntos
Barreira Hematoneural/metabolismo , Células Endoteliais/metabolismo , Miofibroblastos/metabolismo , Nervos Periféricos/metabolismo , Animais , Transporte Biológico , Biomarcadores , Barreira Hematoneural/efeitos dos fármacos , Barreira Hematoneural/patologia , Permeabilidade Capilar , Células Cultivadas , Condutividade Elétrica , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/patologia , Homeostase , Humanos , Miofibroblastos/efeitos dos fármacos , Miofibroblastos/patologia , Nervos Periféricos/efeitos dos fármacos , Nervos Periféricos/patologia , Junções Íntimas/metabolismoRESUMO
Solute and macromolecular transport studies may elucidate nutritional requirements and drug effects in healthy and diseased peripheral nerves. Endoneurial endothelial cells are specialized microvascular cells that form the restrictive blood-nerve barrier (BNB). Primary human endoneurial endothelial cells (pHEndECs) are difficult to isolate, limiting their widespread availability for biomedical research. We developed a simian virus-40 large T-antigen (SV40-LTA) immortalized human BNB cell line via stable transfection of low passage pHEndECs and observed continuous growth in culture for >45 population doublings. As observed with pHEndECs, the immortalized BNB endothelial cells were Ulex Europaeus agglutinin-1-positive and endocytosed low density lipoprotein, but lost von Willebrand factor expression. Glucose transporter-1, P-glycoprotein (P-gp), γ-glutamyl transpeptidase (γ-GT), large neutral amino acid transporter-1 (LAT-1), creatine transporter (CRT), and monocarboxylate transporter-1 (MCT-1) mRNA expression were retained at all passages with loss of alkaline phosphatase (AP) expression after passages 16-20. Compared with an SV40-LTA immortalized human blood-brain barrier endothelial cell line, there was increased γ-GT protein expression, equivalent expression of organic anion transporting polypeptide-C (OATP-C), organic anion transporter 3 (OAT-3), MCT-1, and LAT-1, and reduced expression of AP, CRT, and P-gp by the BNB cell line at passage 20. Further studies demonstrated lower transendothelial electrical resistance (~181 vs. 191 Ω cm(2)), equivalent permeability to fluoresceinated sodium (4.84 vs. 4.39 %), and lower permeability to fluoresceinated high molecular weight (70 kDa) dextran (0.39 vs. 0.52 %) by the BNB cell line. This cell line retained essential molecular and biophysical properties suitable for in vitro peripheral nerve permeability studies.
Assuntos
Barreira Hematoneural/citologia , Técnicas de Cultura de Células/métodos , Linhagem Celular Transformada/citologia , Permeabilidade da Membrana Celular , Células Endoteliais/citologia , Fenômenos Biofísicos , Barreira Hematoencefálica/citologia , Proliferação de Células , Impedância Elétrica , Matriz Extracelular/metabolismo , Humanos , Proteínas de Membrana Transportadoras/metabolismo , Nervos Periféricos/citologiaRESUMO
Several mitogens such as vascular endothelial growth factor (VEGF) have been implicated in mammalian vascular proliferation and repair. However, the molecular mediators of human blood-nerve barrier (BNB) development and specialization are unknown. Primary human endoneurial endothelial cells (pHEndECs) were expanded in vitro and specific mitogen receptors detected by western blot. pHEndECs were cultured with basal medium containing different mitogen concentrations with or without heparin. Non-radioactive cell proliferation, Matrigel(™)-induced angiogenesis and sterile micropipette injury wound healing assays were performed. Proliferation rates, number and total length of induced microvessels, and rate of endothelial cell monolayer wound healing were determined and compared to basal conditions. VEGF-A165 in the presence of heparin, was the most potent inducer of pHEndEC proliferation, angiogenesis, and wound healing in vitro. 1.31 nM VEGF-A165 induced ~110 % increase in cell proliferation relative to basal conditions (â¼51 % without heparin). 2.62 pM VEGF-A165 induced a three-fold increase in mean number of microvessels and 3.9-fold increase in total capillary length/field relative to basal conditions. In addition, 0.26 nM VEGF-A165 induced â¼1.3-fold increased average rate of endothelial wound healing 4-18 h after endothelial monolayer injury, mediated by increased cell migration. VEGF-A165 was the only mitogen capable of complete wound closure, occurring within 30 h following injury via increased cell proliferation. This study demonstrates that VEGF-A165, in the presence of heparin, is a potent inducer of pHEndEC proliferation, angiogenesis, and wound healing in vitro. VEGF-A165 may be an important mitogen necessary for human BNB development and recovery in response to peripheral nerve injury.
Assuntos
Barreira Hematoneural/citologia , Células Endoteliais/citologia , Neovascularização Fisiológica/efeitos dos fármacos , Fator A de Crescimento do Endotélio Vascular/farmacologia , Cicatrização/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Heparina/farmacologia , Humanos , Microvasos/efeitos dos fármacos , Microvasos/metabolismo , Mitógenos/farmacologiaRESUMO
INTRODUCTION: Chemokines and their receptors are important mediators of inflammation. Guillain-Barré syndrome (GBS) is the most common cause of acute paralysis worldwide. Despite current treatments, outcomes are suboptimal. Specific chemokine receptor antagonists have the potential to be efficacious against pathogenic leukocyte trafficking in GBS. METHODS: A 36-year literature review was performed to summarize available data on chemokine expression in GBS and its representative animal model, experimental autoimmune neuritis (EAN). RESULTS: Although there were a few observational human and animal studies demonstrating chemokine ligand/receptor expression in GBS and EAN, in vitro and in vivo functional studies using gene knockouts, neutralizing antibodies, or small molecular antagonists were limited. CCL2-CCR2, CCL5-CCR5, and CXCL10-CXCR3 have been most strongly implicated in EAN and GBS pathogenesis, providing targets for molecular blockade. CONCLUSIONS: Preclinical human in vitro and in vivo EAN studies are needed to evaluate the potential efficacy of chemokine signaling inhibition in GBS.
Assuntos
Quimiocinas/metabolismo , Síndrome de Guillain-Barré/imunologia , Síndrome de Guillain-Barré/metabolismo , Animais , Bases de Dados Factuais/estatística & dados numéricos , HumanosRESUMO
Introduction: Major histocompatibility complex (MHC) class II professional antigen presenting cell-naïve CD4+ T cell interactions via the T-cell receptor complex are necessary for adaptive immunity. MHC class II upregulation in multiple cell types occurs in human autoimmune polyneuropathy patient biopsies, necessitating studies to ascertain cellular signaling pathways required for tissue-specific autoimmunity. Methods: Cryopreserved Guillain-Barré syndrome (GBS) patient sural nerve biopsies and sciatic nerves from the severe murine experimental autoimmune neuritis (sm-EAN) GBS model were studied. Cultured conditional ready MHC Class II antigen A-alpha chain (H2-Aa) embryonic stem cells were used to generate H2-Aa flox/+ C57BL/6 mice. Mice were backcrossed and intercrossed to the SJL background to generate H2-Aa flox/flox SJL mice, bred with hemizygous Tamoxifen-inducible von Willebrand factor Cre recombinase (vWF-iCre/+) SJL mice to generate H2-Aa flox/flox ; vWF-iCre/+ to study microvascular endothelial cell adaptive immune responses. Sm-EAN was induced in adult female SJL Tamoxifen-treated H2-Aa flox/flox ; vWF-iCre/+ mice and H2-Aa flox/flox ; +/+ littermate controls. Neurobehavioral, electrophysiological and histopathological assessments were performed at predefined time points. Results: Endoneurial endothelial cell MHC class II expression was observed in normal and inflamed human and mouse peripheral nerves. Adult female Tamoxifen-treated H2-Aa flox/flox ; vWF-iCre/+ did not develop sm-EAN despite extensive MHC class II expression in lymphoid and non-lymphoid tissues. Discussion: A conditional MHC class II knockout mouse to study cell- and time-dependent adaptive immune responses in vivo is developed. Initial studies show microvascular endothelial cell MHC class II expression is necessary for peripheral nerve specific autoimmunity, as advocated by human in vitro adaptive immunity and ex vivo transplant rejection studies.
RESUMO
Background: Mitochondrial cardiomyopathy (MCM) is an alteration in cardiac structure and function caused by gene mutations or deletions affecting components of the mitochondrial respiratory chain. We report a case of MCM presenting as cardiogenic shock, ultimately requiring left ventricular assist device (LVAD) placement. Case summary: A 35-year-old woman with chronic weakness and non-ischaemic cardiomyopathy, on home dobutamine, was referred to our institution for heart transplantation evaluation. She was admitted to the hospital for suspected cardiogenic shock after laboratory tests revealed a lactate level of 5.4â mmol/L (ref: 0.5-2.2â mmol/L). Her hospital course was complicated by persistently undulating lactate levels (0.2-8.6â mmol/L) that increased with exertion and did not correlate with mixed venous oxygen saturation measurements obtained from a pulmonary artery catheter. Electrodiagnostic testing demonstrated a proximal appendicular and axial myopathy. A left deltoid muscle biopsy was performed that demonstrated evidence of a mitochondrial disease on light and electron microscopy. Muscle genetic testing revealed two large-scale mitochondrial deoxyribonucleic acid sequence deletions, confirming the diagnosis of MCM. She subsequently underwent LVAD placement, which was complicated by significant right ventricular failure requiring early mechanical support. She was ultimately discharged home with chronic inotropic support. Discussion: Mitochondrial cardiomyopathy in adults is a diagnostic and therapeutic challenge. Prompt diagnosis should be made in patients with unknown causes of heart failure via skeletal muscle histopathology guided by electrodiagnostic studies, and targeted genetic testing in affected tissue. Outcomes in adult MCM patients who receive an LVAD are unknown and warrant further investigation.