RESUMEN
Systemic lupus erythematosus is an autoimmune disease that results in immune-mediated damage to kidneys and other organs. We investigated the role of response gene to complement-32 (RGC-32), a proinflammatory and profibrotic mediator induced by TGFß and C5b-9, in nephrotoxic nephritis (NTN), an experimental model that mimics human lupus nephritis. Proteinuria, loss of renal function and kidney histopathology were attenuated in RGC-32 KO NTN mice. RGC-32 KO NTN mice displayed downregulation of the CCL20/CCR6 and CXCL9/CXCR3 ligand/receptor pairs resulting in decreased renal recruitment of IL-17+ and IFNγ+ cells and subsequent decrease in the influx of innate immune cells. RGC-32 deficiency attenuated renal fibrosis as demonstrated by decreased deposition of collagen I, III and fibronectin. Thus, RGC-32 is a unique mediator shared by the Th17 and Th1 dependent proinflammatory and profibrotic pathways and a potential novel therapeutic target in the treatment of immune complex mediated glomerulonephritis such as lupus nephritis.
Asunto(s)
Riñón , Animales , Humanos , Ratones , Modelos Animales de Enfermedad , Fibrosis , Inflamación/inmunología , Riñón/patología , Riñón/inmunología , Nefritis Lúpica/inmunología , Nefritis Lúpica/patología , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas Nucleares , Células TH1/inmunología , Células Th17/inmunologíaRESUMEN
Proliferation of endothelial cells (EC) and smooth muscle cells (SMC) is a critical process in atherosclerosis. Here, we investigated the involvement of sublytic C5b-9 effector Response Gene to Complement 32 (RGC-32) in cell cycle activation, phenotypic switch, and production of extracellular matrix (ECM) in SMC. Overexpression of RGC-32 augmented C5b-9-induced cell cycle activation and proliferation of SMC in an ERK1-dependent manner and silencing of RGC-32 inhibited C5b-9-induced cell cycle activation. C5b-9-induced cell cycle activation also required phosphorylation of RGC-32 at threonine 91. We found that ECM components fibronectin and collagens I-V were expressed by SMC in human aortic atherosclerotic tissue. Silencing of RGC-32 in cultured SMC was followed by a significant reduction in TGF-ß-induced expression of SMC differentiation markers myocardin, SM22 and α-SMA, and that of collagens I, IV and V. These data suggest that RGC-32 participates in both sublytic C5b-9-induced cell cycle activation and TGF-ß-induced ECM production.
Asunto(s)
Aterosclerosis , Proteínas de Ciclo Celular , Complejo de Ataque a Membrana del Sistema Complemento , Proteínas Musculares , Proteínas del Tejido Nervioso , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Células Cultivadas , Complejo de Ataque a Membrana del Sistema Complemento/metabolismo , Proteínas del Sistema Complemento , Células Endoteliales , Humanos , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Miocitos del Músculo Liso/metabolismo , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Factor de Crecimiento Transformador betaRESUMEN
In this study, we investigated the role of JNK and phospho-Bcl-2 as possible biomarkers of multiple sclerosis (MS) relapse and of glatiramer acetate (GA) therapeutic response in relapsing-remitting MS patients. We enrolled a cohort of 15 GA-treated patients and measured the expression of JNK1, JNK2, phospho-JNK and phospho-Bcl-2 through Western blotting of lysates from peripheral blood mononuclear cells collected at 0, 3, 6, and 12â¯months after initiating GA therapy. We found significantly higher levels of JNK1 p54 and JNK2 p54 and significantly lower levels of p-Bcl-2 in relapse patients and in GA non-responders. By using receiver operating characteristic analysis, we found that the probability of accurately detecting relapse and response to GA was: 92% and 75.5%, respectively, for JNK1 p54 and 86% and 94.6%, respectively, for p-Bcl-2. Our data suggest that JNK1 and p-Bcl-2 could serve as potential biomarkers for MS relapse and the therapeutic response to GA.
Asunto(s)
Biomarcadores Farmacológicos/metabolismo , Biomarcadores/metabolismo , Proteína Quinasa 8 Activada por Mitógenos/metabolismo , Proteína Quinasa 9 Activada por Mitógenos/metabolismo , Esclerosis Múltiple Recurrente-Remitente/diagnóstico , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Adolescente , Adulto , Anciano , Estudios de Cohortes , Progresión de la Enfermedad , Femenino , Regulación de la Expresión Génica , Acetato de Glatiramer/uso terapéutico , Humanos , Masculino , Persona de Mediana Edad , Esclerosis Múltiple Recurrente-Remitente/tratamiento farmacológico , Fosforilación , Valor Predictivo de las Pruebas , Adulto JovenRESUMEN
There is increasing awareness that in addition to the metabolic crisis of diabetic ketoacidosis (DKA) caused by severe insulin deficiency, the immune inflammatory response is likely an active multicomponent participant in both the acute and chronic insults of this medical crisis, with strong evidence of activation for both the cytokine and complement system. Recent studies report that the matrix metalloproteinase enzymes and their inhibitors are systemically activated in young Type 1 diabetes mellitus (T1D) patients during DKA and speculate on their involvement in blood-brain barrier (BBB) disruption. Based on our previous studies, we address the question if matrix metalloproteinase 9 (MMP9) is expressed in the brain in the fatal brain edema (BE) of DKA. Our data show significant expression of MMP9 on the cells present in brain intravascular areas. The presence of MMP9 in intravascular cells and that of MMP+ cells seen passing the BBB indicates a possible role in tight junction protein disruption of the BBB, possibly leading to neurological complications including BE. We have also shown that MMP9 is expressed on neurons in the hippocampal areas of both BE/DKA cases investigated, while expression of tissue inhibitor of metalloproteinases 1 (TIMP1) was reduced in the same areas. We can speculate that intraneuronal MMP9 can be a sign of neurodegeneration. Further studies are necessary to determine the role of MMP9 in the pathogenesis of the neurologic catastrophe of the brain edema of DKA. Inhibition of MMP9 expression might be helpful in preserving neuronal function and BBB integrity during DKA.
Asunto(s)
Cetoacidosis Diabética/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , Adolescente , Barrera Hematoencefálica/metabolismo , Encéfalo/metabolismo , Edema Encefálico/genética , Edema Encefálico/metabolismo , Cetoacidosis Diabética/mortalidad , Femenino , Hipocampo/metabolismo , Humanos , Metaloproteinasas de la Matriz/metabolismo , Neuronas/metabolismo , Uniones Estrechas/metabolismo , Inhibidor Tisular de Metaloproteinasa-1/genética , Inhibidor Tisular de Metaloproteinasa-1/metabolismo , Transcriptoma/genéticaRESUMEN
Th17 cells play a critical role in autoimmune diseases, including multiple sclerosis and its animal model, experimental autoimmune encephalomyelitis. Response gene to complement (RGC)-32 is a cell cycle regulator and a downstream target of TGF-ß that mediates its profibrotic activity. In this study, we report that RGC-32 is preferentially upregulated during Th17 cell differentiation. RGC-32-/- mice have normal Th1, Th2, and regulatory T cell differentiation but show defective Th17 differentiation in vitro. The impaired Th17 differentiation is associated with defects in IFN regulatory factor 4, B cell-activating transcription factor, retinoic acid-related orphan receptor γt, and SMAD2 activation. In vivo, RGC-32-/- mice display an attenuated experimental autoimmune encephalomyelitis phenotype accompanied by decreased CNS inflammation and reduced frequency of IL-17- and GM-CSF-producing CD4+ T cells. Collectively, our results identify RGC-32 as a novel regulator of Th17 cell differentiation in vitro and in vivo and suggest that RGC-32 is a potential therapeutic target in multiple sclerosis and other Th17-mediated autoimmune diseases.
Asunto(s)
Diferenciación Celular/inmunología , Encefalomielitis Autoinmune Experimental/inmunología , Regulación de la Expresión Génica , Proteínas Nucleares/genética , Proteínas Nucleares/fisiología , Células Th17/fisiología , Animales , Diferenciación Celular/efectos de los fármacos , Sistema Nervioso Central/inmunología , Sistema Nervioso Central/fisiopatología , Factor Estimulante de Colonias de Granulocitos y Macrófagos/biosíntesis , Factor Estimulante de Colonias de Granulocitos y Macrófagos/inmunología , Factores Reguladores del Interferón/genética , Factores Reguladores del Interferón/metabolismo , Activación de Linfocitos , Ratones , Ratones Endogámicos C57BL , Proteínas Nucleares/deficiencia , Proteínas Nucleares/farmacología , Receptores de Ácido Retinoico/genética , Receptores de Ácido Retinoico/metabolismo , Células TH1/inmunología , Células Th17/inmunología , Células Th17/patologíaRESUMEN
We have previously shown that SIRT1 mRNA expression was significantly lower in relapsing MS patients compared to those in remission. Our goal was to longitudinally investigate the role of active, phosphorylated SIRT1 (p-SIRT1) as a potential biomarker of relapse and predictor for response to glatiramer acetate (GA) treatment in patients with relapsing remitting multiple sclerosis (MS). We also want to investigate the downstream effects of SIRT1 activation by measuring the trimethylation of histone 3 at lysine 9 (H3K9me3). A cohort of 15 GA-treated patients was clinically monitored using the Expanded Disability Status Scale (EDSS) and peripheral blood mononuclear cells (PBMCs) were collected at 0, 3, 6, and 12â¯months after initiation of the therapy. P-SIRT1 and H3K9me3 levels were assayed by Western blotting using specific antibodies. Statistically significant lower levels of p-SIRT1 protein (pâ¯<â¯0.0001) and H3K9me3 (pâ¯=â¯0.001) were found during relapses when compared to stable MS patients. Non-responders to GA treatment were defined as patients who exhibited at least two relapses following initiation of GA treatment. Statistically significant lower levels of p-SIRT1 protein (pâ¯=â¯0.02) and H3K9me3 (pâ¯=â¯0.004) were found in GA non-responders compared to responders. Using receiver operating characteristic analysis, area under the curve (AUC) for p-SIRT1 was 77% (pâ¯=â¯0.007) and for H3K9me3 was 81% (pâ¯=â¯0.002) for prediction of relapse. For predicting responsiveness to GA treatment, AUC was 75% (Pâ¯=â¯0.01) for H3K9me3. Our data suggest that p-SIRT1 and H3K9me3 could serve as potential biomarkers for MS relapse. In addition, H3K9me3 could serve as possible biomarker to predict response to GA treatment.
Asunto(s)
Acetato de Glatiramer/uso terapéutico , Esclerosis Múltiple Recurrente-Remitente/tratamiento farmacológico , Sirtuina 1/metabolismo , Adulto , Biomarcadores Farmacológicos/metabolismo , Estudios de Cohortes , Metilación de ADN , Femenino , Histonas/genética , Histonas/metabolismo , Humanos , Leucocitos Mononucleares/metabolismo , Estudios Longitudinales , Masculino , Persona de Mediana Edad , Esclerosis Múltiple Recurrente-Remitente/enzimología , Esclerosis Múltiple Recurrente-Remitente/genética , Esclerosis Múltiple Recurrente-Remitente/metabolismo , Fosforilación , Recurrencia , Sirtuina 1/genéticaRESUMEN
Due to the limited data on diabetic ketoacidosis and brain edema (DKA/BE) in children/adolescents and the lack of recent data on adults with type 1 diabetes (T1D), we addressed the question of whether neuroinflammation was present in the fatal DKA of adults. We performed immunohistochemistry (IHC) studies on the brains of two young adults with T1D and fatal DKA and compared them with two teenagers with poorly controlled diabetes and fatal DKA. C5b-9, the membrane attack complex (MAC) had significantly greater deposits in the grey and white matter of the teenagers than the young adults (p=0.03). CD59, a MAC assembly inhibitory protein was absent, possibly suppressed by the hyperglycemia in the teenagers but was expressed in the young adults despite comparable average levels of hyperglycemia. The receptor for advanced glycation end products (RAGE) had an average expression in the young adults significantly greater than in the teenagers (p=0.02). The autophagy marker Light Chain 3 (LC3) A/B was the predominant form of programmed cell death (PCD) in the teenage brains. The young adults had high expressions of both LC3A/B and TUNEL, an apoptotic cell marker for DNA fragmentation. BE was present in the newly diagnosed young adult with hyperglycemic hyperosmolar DKA and also in the two teenagers. Our data indicate that significant differences in neuroinflammatory components, initiated by the dysregulation of DKA and interrelated metabolic and immunologic milieu, are likely present in the brains of fatal DKA of teenagers when compared with young adults.
Asunto(s)
Biomarcadores/metabolismo , Diabetes Mellitus Tipo 1/genética , Cetoacidosis Diabética/genética , Inflamación Neurogénica/genética , Adolescente , Adulto , Autofagia , Encéfalo/fisiopatología , Edema Encefálico/diagnóstico , Edema Encefálico/etiología , Edema Encefálico/genética , Antígenos CD59/genética , Antígenos CD59/metabolismo , Fragmentación del ADN , Diabetes Mellitus Tipo 1/complicaciones , Cetoacidosis Diabética/complicaciones , Regulación de la Expresión Génica , Humanos , Inmunohistoquímica , Etiquetado Corte-Fin in Situ , Mediadores de Inflamación/metabolismo , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Inflamación Neurogénica/etiología , Adulto JovenRESUMEN
SIRT1, a NAD dependent histone and protein deacetylase, is a member of the histone deacetylase class III family. We previously showed that SIRT1 mRNA expression is significantly lower in peripheral blood mononuclear cells (PBMCs) of multiple sclerosis (MS) patients during relapses than in stable patients. We have now investigated SIRT1 as a possible biomarker to predict relapse as well as responsiveness to glatiramer acetate (GA) treatment in relapsing-remitting MS (RRMS) patients. Over the course of 2years, a cohort of 15 GA-treated RRMS patients were clinically monitored using the Expanded Disability Status Scale and assessed for MS relapses. Blood samples collected from MS patients were analyzed for levels of SIRT1 and histone H3 lysine 9 (H3K9) acetylation and dimethylation. During relapses, MS patients had a lower expression of SIRT1 mRNA than did stable MS patients. In addition, there was a significant decrease in H3K9 dimethylation (H3K9me2) during relapses in MS patients when compared to stable patients (p=0.01). Responders to GA treatment had significantly higher SIRT1 mRNA (p=0.01) and H3K9me2 levels than did non-responders (p=0.018). Receiver operating characteristic analysis was used to assess the predictive power of SIRT1 and H3K9me2 as putative biomarkers: for SIRT1 mRNA, the predictive value for responsiveness to GA treatment was 70% (p=0.04) and for H3K9me2 was 71% (p=0.03). Our data suggest that SIRT1 and H3K9me2 could serve as potential biomarkers for evaluating patients' responsiveness to GA therapy in order to help guide treatment decisions in MS.
Asunto(s)
Acetato de Glatiramer/uso terapéutico , Histonas/metabolismo , Esclerosis Múltiple Recurrente-Remitente/tratamiento farmacológico , Esclerosis Múltiple Recurrente-Remitente/genética , Sirtuina 1/metabolismo , Acetilación , Adulto , Biomarcadores/metabolismo , Femenino , Regulación de la Expresión Génica , Humanos , Masculino , Persona de Mediana Edad , ARN Mensajero/genética , ARN Mensajero/metabolismo , Recurrencia , Sirtuina 1/genética , Adulto JovenRESUMEN
The complement system is an important player in the development of atherosclerosis. Previously reported as a cell cycle regulator, RGC-32 is an essential effector of the terminal complement complex, C5b-9. In this study, our aims were to determine the expression of RGC-32 in the human atherosclerotic arterial wall and to delineate the mechanisms through which RGC-32 affects C5b-9-induced endothelial cell proliferation and migration. We now demonstrate that RGC-32 is expressed in human aortic atherosclerotic wall and that RGC-32 expression increases with the progression of atherosclerosis. Furthermore, silencing of RGC-32 expression abolished C5b-9-induced human aortic endothelial cell (HAEC) proliferation and migration. Of the 279 genes differentially expressed in HAECs after RGC-32 silencing, the genes involved in cell adhesion and cell cycle activation were significantly regulated by RGC-32. RGC-32 silencing caused a significant reduction in the expression of cyclin D1, cyclin D3, Akt, ROCK1, Rho GDP dissociation inhibitor alpha and profilin. These data suggest that RGC-32 mediates HAEC migration through the regulation of RhoA and ROCK1 expression and is involved in actin cytoskeletal organization. Thus, RGC-32 has promising therapeutic potential with regard to angiogenesis and atherosclerosis.
Asunto(s)
Aorta/patología , Aterosclerosis/patología , Proteínas de Ciclo Celular/metabolismo , Movimiento Celular , Complejo de Ataque a Membrana del Sistema Complemento/metabolismo , Proteínas Musculares/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Anciano , Anciano de 80 o más Años , Aorta/metabolismo , Aterosclerosis/genética , Western Blotting , Proliferación Celular , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Femenino , Silenciador del Gen , Humanos , Inmunohistoquímica , Masculino , Persona de Mediana Edad , Mitosis , Miocitos del Músculo Liso/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Transcripción GenéticaRESUMEN
Currently there is critical need for the identification of reliable biomarkers to help guide clinical management of multiple sclerosis (MS) patients. We investigated the combined roles of Response Gene to Complement 32 (RGC-32), FasL, CDC2, AKT, and IL-21 as possible biomarkers of relapse and response to glatiramer acetate (GA) treatment in relapsing-remitting MS (RRMS) patients. Over the course of 2 years, a cohort of 15 GA-treated RRMS patients was clinically monitored and peripheral blood mononuclear cells (PBMCs) were collected at 0, 3, 6, and 12 months. Target gene mRNA expression was measured in patients' isolated PBMCs by real-time qRT-PCR. Compared to stable MS patients, those with acute relapses exhibited decreased expression of RGC-32 (p<0.0001) and FasL (p<0.0001), increased expression of IL-21 (p=0.04), but no change in CDC2 or AKT. Compared to non-responders, responders to GA treatment showed increased expression of RGC-32 (p<0.0001) and FasL (p<0.0001), and decreased expression of IL-21 (p=0.02). Receiver operating characteristic (ROC) analysis was used to assess the predictive accuracy of each putative biomarker. The probability of accurately detecting relapse was 90% for RGC-32, 88% for FasL, and 75% for IL-21. The probability of accurately detecting response to GA was 85% for RGC-32, 90% for FasL, and 85% for IL-21. Our data suggest that RGC-32, FasL, and IL-21 could serve as potential biomarkers for the detection of MS relapse and response to GA therapy.
Asunto(s)
Proteínas de Ciclo Celular/genética , Acetato de Glatiramer/uso terapéutico , Leucocitos Mononucleares/metabolismo , Esclerosis Múltiple/tratamiento farmacológico , Esclerosis Múltiple/genética , Proteínas Musculares/genética , Proteínas del Tejido Nervioso/genética , Adulto , Biomarcadores/análisis , Biomarcadores/metabolismo , Proteínas de Ciclo Celular/metabolismo , Femenino , Humanos , Interleucinas/metabolismo , Masculino , Persona de Mediana Edad , Proteínas Musculares/metabolismo , Proteínas del Tejido Nervioso/metabolismo , RecurrenciaRESUMEN
Regular assessment of disease activity in relapsing-remitting multiple sclerosis (RRMS) is required to optimize clinical outcomes. Biomarkers can be a valuable tool for measuring disease activity in multiple sclerosis (MS) if they reflect the pathological processes underlying MS pathogenicity. In this pilot study, we combined multiple biomarkers previously analyzed in RRMS patients into an MS disease activity (MSDA) score to evaluate their ability to predict relapses and treatment response to glatiramer acetate (GA). Response Gene to Complement 32 (RGC-32), FasL, IL-21, SIRT1, phosphorylated SIRT1 (p-SIRT1), and JNK1 p54 levels were used to generate cut-off values for each biomarker. Any value below the cutoff for RGC-32, FasL SIRT1, or p-SIRT1 or above the cutoff for IL-21 or JNK1 p54 was given a +1 value, indicating relapse or lack of response to GA. Any value above the cutoff value for RGC-32, FasL, SIRT1, p-SIRT1 or below that for IL-21 or JNK1 p54 was given a -1 value, indicating clinical stability or response to GA. An MSDA score above +1 indicated a relapse or lack of response to treatment. An MSDA score below -1 indicated clinical stability or response to treatment. Our results showed that the MSDA scores generated using either four or six biomarkers had a higher sensitivity and specificity and significantly correlated with the expanded disability status scale. Although these results suggest that the MSDA test can be useful for monitoring therapeutic response to biologic agents and assessing clinically challenging situations, the present findings need to be confirmed in larger studies.
Asunto(s)
Biomarcadores , Acetato de Glatiramer , Sirtuina 1 , Humanos , Masculino , Adulto , Femenino , Sirtuina 1/metabolismo , Acetato de Glatiramer/uso terapéutico , Persona de Mediana Edad , Esclerosis Múltiple Recurrente-Remitente/tratamiento farmacológico , Esclerosis Múltiple Recurrente-Remitente/diagnóstico , Proteína Ligando Fas/metabolismo , Resultado del Tratamiento , Proyectos Piloto , Proteína Quinasa 8 Activada por Mitógenos/metabolismo , Interleucinas , Esclerosis Múltiple/tratamiento farmacológico , Esclerosis Múltiple/diagnóstico , Índice de Severidad de la Enfermedad , Inmunosupresores/uso terapéuticoRESUMEN
The brains of COVID-19 patients are affected by the SARS-CoV-2 virus, and these effects may contribute to several COVID-19 sequelae, including cognitive dysfunction (termed "long COVID" by some researchers). Recent advances concerning the role of neuroinflammation and the consequences for brain function are reviewed in this manuscript. Studies have shown that respiratory SARS-CoV-2 infection in mice and humans is associated with selective microglial reactivity in the white matter, persistently impaired hippocampal neurogenesis, a decrease in the number of oligodendrocytes, and myelin loss. Brain MRI studies have revealed a greater reduction in grey matter thickness in the orbitofrontal cortex and parahippocampal gyrus, associated with a greater reduction in global brain size, in those with SARS-CoV-2 and a greater cognitive decline. COVID-19 can directly infect endothelial cells of the brain, potentially promoting clot formation and stroke; complement C3 seems to play a major role in this process. As compared to controls, the brain tissue of patients who died from COVID-19 have shown a significant increase in the extravasation of fibrinogen, indicating leakage in the blood-brain barrier; furthermore, recent studies have documented the presence of IgG, IgM, C1q, C4d, and C5b-9 deposits in the brain tissue of COVID-19 patients. These data suggest an activation of the classical complement pathway and an immune-mediated injury to the endothelial cells. These findings implicate both the classical and alternative complement pathways, and they indicate that C3b and the C5b-9 terminal complement complex (membrane attack complex, MAC) are acting in concert with neuroinflammatory and immune factors to contribute to the neurological sequelae seen in patients with COVID.
Asunto(s)
COVID-19 , Complejo de Ataque a Membrana del Sistema Complemento , Humanos , Ratones , Animales , Complejo de Ataque a Membrana del Sistema Complemento/metabolismo , Células Endoteliales/metabolismo , SARS-CoV-2/metabolismo , Encéfalo/metabolismoRESUMEN
Recent advances in understanding the pathogenesis of multiple sclerosis (MS) have brought into the spotlight the major role played by reactive astrocytes in this condition. Response Gene to Complement (RGC)-32 is a gene induced by complement activation, growth factors, and cytokines, notably transforming growth factor ß, that is involved in the modulation of processes such as angiogenesis, fibrosis, cell migration, and cell differentiation. Studies have uncovered the crucial role that RGC-32 plays in promoting the differentiation of Th17 cells, a subtype of CD4+ T lymphocytes with an important role in MS and its murine model, experimental autoimmune encephalomyelitis. The latest data have also shown that RGC-32 is involved in regulating major transcriptomic changes in astrocytes and in favoring the synthesis and secretion of extracellular matrix components, growth factors, axonal growth molecules, and pro-astrogliogenic molecules. These results suggest that RGC-32 plays a major role in driving reactive astrocytosis and the generation of astrocytes from radial glia precursors. In this review, we summarize recent advances in understanding how RGC-32 regulates the behavior of Th17 cells and astrocytes in neuroinflammation, providing insight into its role as a potential new biomarker and therapeutic target.
Asunto(s)
Proteínas de Ciclo Celular , Esclerosis Múltiple , Proteínas Musculares , Proteínas del Tejido Nervioso , Animales , Biomarcadores , Proteínas de Ciclo Celular/genética , Proteínas del Sistema Complemento , Citocinas , Humanos , Ratones , Proteínas Musculares/genética , Proteínas del Tejido Nervioso/genética , Enfermedades Neuroinflamatorias , Proteínas Nucleares/genética , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
Response Gene to Complement 32 (RGC-32) is an important mediator of the TGF-ß signaling pathway, and an increasing amount of evidence implicates this protein in regulating astrocyte biology. We showed recently that spinal cord astrocytes in mice lacking RGC-32 display an immature phenotype reminiscent of progenitors and radial glia, with an overall elongated morphology, increased proliferative capacity, and increased expression of progenitor markers when compared to their wild-type (WT) counterparts that make them incapable of undergoing reactive changes during the acute phase of experimental autoimmune encephalomyelitis (EAE). Here, in order to decipher the molecular networks underlying RGC-32's ability to regulate astrocytic maturation and reactivity, we performed next-generation sequencing of RNA from WT and RGC-32 knockout (KO) neonatal mouse brain astrocytes, either unstimulated or stimulated with the pleiotropic cytokine TGF-ß. Pathway enrichment analysis showed that RGC-32 is critical for the TGF-ß-induced up-regulation of transcripts encoding proteins involved in brain development and tissue remodeling, such as axonal guidance molecules, transcription factors, extracellular matrix (ECM)-related proteins, and proteoglycans. Our next-generation sequencing of RNA analysis also demonstrated that a lack of RGC-32 results in a significant induction of WD repeat and FYVE domain-containing protein 1 (Wdfy1) and stanniocalcin-1 (Stc1). Immunohistochemical analysis of spinal cords isolated from normal adult mice and mice with EAE at the peak of disease showed that RGC-32 is necessary for the in vivo expression of ephrin receptor type A7 in reactive astrocytes, and that the lack of RGC-32 results in a higher number of homeodomain-only protein homeobox (HOPX)+ and CD133+ radial glia cells. Collectively, these findings suggest that RGC-32 plays a major role in modulating the transcriptomic changes in astrocytes that ultimately lead to molecular programs involved in astrocytic differentiation and reactive changes during neuroinflammation.
Asunto(s)
Astrocitos/metabolismo , Gliosis/genética , Enfermedades Neuroinflamatorias/genética , Proteínas Nucleares/fisiología , Transcriptoma , Animales , Orientación del Axón/genética , Encéfalo/patología , Encefalomielitis Autoinmune Experimental/genética , Encefalomielitis Autoinmune Experimental/inmunología , Encefalomielitis Autoinmune Experimental/patología , Femenino , Regulación de la Expresión Génica , Ontología de Genes , Redes Reguladoras de Genes , Gliosis/etiología , Gliosis/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas del Tejido Nervioso/biosíntesis , Proteínas del Tejido Nervioso/genética , Células-Madre Neurales/metabolismo , Neurogénesis , Enfermedades Neuroinflamatorias/metabolismo , Proteínas Nucleares/deficiencia , Organismos Libres de Patógenos Específicos , Médula Espinal/patologíaRESUMEN
Sublytic levels of C5b-9 increase the survival of oligodendrocytes (OLGs) and induce the cell cycle. We have previously observed that SIRT1 co-localizes with surviving OLGs in multiple sclerosis (MS) plaques, but it is not yet known whether SIRT1 is involved in OLGs survival after exposure to sublytic C5b-9. We have now investigated the role of SIRT1 in OLGs differentiation and the effect of sublytic levels of C5b-9 on SIRT1 and phosphorylated-SIRT1 (Ser27) expression. We also examined the downstream effects of SIRT1 by measuring histone H3 lysine 9 trimethylation (H3K9me3) and the expression of cyclin D1 as a marker of cell cycle activation. OLG progenitor cells (OPCs) purified from the brain of rat pups were differentiated in vitro and treated with sublytic C5b-9 or C5b6. To investigate the signaling pathway activated by C5b-9 and required for SIRT1 expression, we pretreated OLGs with a c-jun antisense oligonucleotide, a phosphoinositide 3-kinase (PI3K) inhibitor (LY294002), and a protein kinase C (PKC) inhibitor (H7). Our data show a significant reduction in phospho-SIRT1 and SIRT1 expression during OPCs differentiation, associated with a decrease in H3K9me3 and a peak of cyclin D1 expression in the first 24 h. Stimulation of OLGs with sublytic C5b-9 resulted in an increase in the expression of SIRT1 and phospho-SIRT1, H3K9me3, cyclin D1 and decreased expression of myelin-specific genes. C5b-9-stimulated SIRT1 expression was significantly reduced after pretreatment with c-jun antisense oligonucleotide, H7 or LY294002. Inhibition of SIRT1 with sirtinol also abolished C5b-9-induced DNA synthesis. Taken together, these data show that induction of SIRT1 expression by C5b-9 is required for cell cycle activation and is mediated through multiple signaling pathways.
Asunto(s)
Complejo de Ataque a Membrana del Sistema Complemento/farmacología , Oligodendroglía/efectos de los fármacos , Sirtuina 1/fisiología , Animales , Ciclo Celular/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Vaina de Mielina/efectos de los fármacos , Oligodendroglía/fisiología , Fosfatidilinositol 3-Quinasas/fisiología , Proteína Quinasa C/fisiología , Ratas , Ratas Sprague-DawleyRESUMEN
Astrocytes are increasingly recognized as critical contributors to multiple sclerosis pathogenesis. We have previously shown that lack of Response Gene to Complement 32 (RGC-32) alters astrocyte morphology in the spinal cord at the peak of experimental autoimmune encephalomyelitis (EAE), suggesting a role for RGC-32 in astrocyte differentiation. In this study, we analyzed the expression and distribution of astrocytes and astrocyte progenitors by immunohistochemistry in spinal cords of wild-type (WT) and RGC-32-knockout (KO) mice with EAE and of normal adult mice. Our analysis showed that during acute EAE, WT astrocytes had a reactive morphology and increased GFAP expression, whereas RGC-32 KO astrocytes had a morphology similar to that of radial glia and an increased expression of progenitor markers such as vimentin and fatty acid binding protein 7 (FABP7). In control mice, GFAP expression and astrocyte density were also significantly higher in the WT group, whereas the number of vimentin and FABP7-positive radial glia was significantly higher in the RGC-32 KO group. In vitro studies on cultured neonatal astrocytes from WT and RGC-32 KO mice showed that RGC-32 regulates a complex array of molecular networks pertaining to signal transduction, growth factor expression and secretion, and extracellular matrix (ECM) remodeling. Among the most differentially expressed factors were insulin-like growth factor 1 (IGF1), insulin-like growth factor binding proteins (IGFBPs), and connective tissue growth factor (CTGF); their expression was downregulated in RGC-32-depleted astrocytes. The nuclear translocation of STAT3, a transcription factor critical for astrogliogenesis and driving glial scar formation, was also impaired after RGC-32 silencing. Taken together, these data suggest that RGC-32 is an important regulator of astrocyte differentiation during EAE and that in the absence of RGC-32, astrocytes are unable to fully mature and become reactive astrocytes.
Asunto(s)
Astrocitos/metabolismo , Proliferación Celular , Encefalomielitis Autoinmune Experimental/metabolismo , Proteínas Nucleares/metabolismo , Médula Espinal/metabolismo , Animales , Astrocitos/patología , Diferenciación Celular , Movimiento Celular , Células Cultivadas , Encefalomielitis Autoinmune Experimental/genética , Encefalomielitis Autoinmune Experimental/patología , Proteína de Unión a los Ácidos Grasos 7/metabolismo , Femenino , Proteína Ácida Fibrilar de la Glía/metabolismo , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas Nucleares/genética , Fenotipo , Ratas Sprague-Dawley , Transducción de Señal , Médula Espinal/patología , Vimentina/metabolismoRESUMEN
Seric biomarkers have been tested in a large number of studies on traumatic brain injuries (TBI) patients in order to predict severity, especially related to the short-term outcome. However, TBI patients have a high risk of developing long-term complications such as physical disability, cognitive impairment, psychiatric pathology, epilepsy, and others. The aim of this study was to assess the correlation between protein biomarkers S100 and neuron-specific enolase (NSE) and neurocognitive status at 10- and 90-days post-injury. Both biomarkers were tested in the first 4h and after 72h post-injury in 62 patients with moderate-severe TBI. The patients were evaluated by a series of neurocognitive tests: Early Rehabilitation Barthel Index (ERBI), Glasgow Outcome Scale-Extended (GOSE), The Mini-Mental State Examination (MMSE), Processing Speed Index (PSI), and Stroop Test, at 10 and 90 days post-injury and supplementary by the Hospital Anxiety and Depression Scale at 90 days. For evaluating the whole neurocognitive status instead of every scale separately, we used Structural Equation Modeling (SEM), while for anxiety and depressive symptoms, we used multiple regression analyses. SEM showed that NSE values at 4 hours were significant predictors of the cognitive status at 10 (p=0.034) and 90 days (p= 0.023). Also, there were found significant correlations between NSE at 4h and the anxiety level. This study demonstrated a significant correlation between NSE at 4h and short and medium-term neuropsychological outcomes, which recommends using this biomarker for selecting patients with a higher risk of cognitive dysfunction.
Asunto(s)
Lesiones Traumáticas del Encéfalo/sangre , Lesiones Traumáticas del Encéfalo/fisiopatología , Cognición , Fosfopiruvato Hidratasa/sangre , Proteínas S100/sangre , Adulto , Biomarcadores/sangre , Emociones , Femenino , Humanos , Masculino , Persona de Mediana Edad , Análisis de Regresión , Adulto JovenRESUMEN
Hydraulic fracturing in shale/tight gas reservoirs creates fracture network systems that can intersect pre-existing subsurface flow pathways, e.g. fractures, faults or abandoned wells. This way, hydraulic fracturing operations could pose environmental risks to shallow groundwater systems. This paper explores the long-term (>â¯30â¯years) flow and transport of fracturing fluids into overburden layers and groundwater aquifers through a leaky abandoned well, using the geological setting of North German Basin as a case study. A three-dimensional model consisting of 15 sedimentary layers with three hydrostratigraphic units representing the hydrocarbon reservoir, overburden, and the aquifer is built. The model considers one perforation location at the first section of the horizontal part of the well, and a discrete hydraulic fracture intersecting an abandoned well. A sensitivity analysis is carried out to quantify and understand the influence of a broad spectrum of field possibilities (reservoir properties, overburden properties, abandoned well properties and its proximity to hydraulic fractures) on the flow of fracturing fluid to shallower permeable strata. The model results suggest the spatial properties of the abandoned well as well as its distance from the hydraulic fracture are the most important factors influencing the vertical flow of fracturing fluid. It is observed that even for various field set-tings, only a limited amount of fracturing fluid can reach the aquifer in a long-term period.
Asunto(s)
Agua Subterránea , Fracking Hidráulico , Gas Natural , Yacimiento de Petróleo y Gas , Pozos de AguaRESUMEN
The complement system represents an effective arsenal of innate immunity as well as an interface between innate and adaptive immunity. Activation of the complement system culminates with the assembly of the C5b-9 terminal complement complex on cell membranes, inducing target cell lysis. Translation of this sequence of events into a malignant setting has traditionally afforded C5b-9 a strict antitumoral role, in synergy with antibody-dependent tumor cytolysis. However, in recent decades, a plethora of evidence has revised this view, highlighting the tumor-promoting properties of C5b-9. Sublytic C5b-9 induces cell cycle progression by activating signal transduction pathways (e.g., Gi protein/ phosphatidylinositol 3-kinase (PI3K)/Akt kinase and Ras/Raf1/ERK1) and modulating the activation of cancer-related transcription factors, while shielding malignant cells from apoptosis. C5b-9 also induces Response Gene to Complement (RGC)-32, a gene that contributes to cell cycle regulation by activating the Akt and CDC2 kinases. RGC-32 is expressed by tumor cells and plays a dual role in cancer, functioning as either a tumor promoter by endorsing malignancy initiation, progression, invasion, metastasis, and angiogenesis, or as a tumor suppressor. In this review, we present recent data describing the versatile, multifaceted roles of C5b-9 and its effector, RGC-32, in cancer.