RESUMO
IFNγ signaling in endothelial (EC) and smooth muscle cells (SMC) is a key culprit of pathologic vascular remodeling. The impact of NF-κB inhibitory protein A20 on IFNγ signaling in vascular cells remains unknown. In gain- and loss-of-function studies, A20 inversely regulated expression of IFNγ-induced atherogenic genes in human EC and SMC by modulating STAT1 transcription. In vivo, inadequate A20 expression in A20 heterozygote mice aggravated intimal hyperplasia following partial carotid artery ligation. This outcome uniquely associated with increased levels of Stat1 and super-induction of Ifnγ-dependent genes. Transcriptome analysis of the aortic media from A20 heterozygote versus wild-type mice revealed increased basal Ifnß signaling as the likely cause for higher Stat1 transcription. We confirmed higher basal IFNß levels in A20-silenced human SMC and showed that neutralization or knockdown of IFNß abrogates heightened STAT1 levels in these cells. Upstream of IFNß, A20-silenced EC and SMC demonstrated higher levels of phosphorylated/activated TANK-binding kinase-1 (TBK1), a regulator of IFNß transcription. This suggested that A20 knockdown increased STAT1 transcription by enhancing TBK1 activation and subsequently basal IFNß levels. Altogether, these results uncover A20 as a key physiologic regulator of atherogenic IFNγ/STAT1 signaling. This novel function of A20 added to its ability to inhibit nuclear factor-κB (NF-κB) activation solidifies its promise as an ideal therapeutic candidate for treatment and prevention of vascular diseases. In light of recently discovered A20/TNFAIP3 (TNFα-induced protein 3) single nucleotide polymorphisms that impart lower A20 expression or function, these results also qualify A20 as a reliable clinical biomarker for vascular risk assessment.
Assuntos
Aterosclerose/metabolismo , Cisteína Endopeptidases/fisiologia , Proteínas de Ligação a DNA/metabolismo , Interferon beta/metabolismo , Interferon gama/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Músculo Liso Vascular/metabolismo , Proteínas Nucleares/metabolismo , Animais , Anti-Inflamatórios/química , Aorta/patologia , Movimento Celular , Constrição Patológica/metabolismo , Cisteína Endopeptidases/metabolismo , Humanos , Inflamação , Camundongos , Fosforilação , Polimorfismo de Nucleotídeo Único , RNA Interferente Pequeno/metabolismo , Fator de Transcrição STAT1/metabolismo , Transdução de Sinais , Proteína 3 Induzida por Fator de Necrose Tumoral alfa , Células U937RESUMO
A1/Bfl-1 is a NF-κB dependent, anti-apoptotic Bcl-2 family member that contains four Bcl-2 homology domains (BH) and an amphipathic C-terminal domain, and is expressed in endothelial cells (EC). Based on NF-κB reporter assays in bovine aortic EC, we have previously demonstrated that A1, like Bcl-2 and Bcl-xL, inhibits NF-κB activation. These results, however, do not fully translate when evaluating the cell's own NF-κB machinery in human EC overexpressing A1 by means of recombinant adenovirus (rAd.) mediated gene transfer. Indeed, overexpression of full-length A1 in human umbilical vein EC (HUVEC), and human dermal microvascular EC (HDMEC) failed to inhibit NF-κB activation. However, overexpression of a mutant lacking the C-terminal domain of A1 (A1ΔC) demonstrated a potent NF-κB inhibitory effect in these cells. Disparate effects of A1 and A1ΔC on NF-κB inhibition in human EC correlated with mitochondrial (A1) versus non-mitochondrial (A1ΔC) localization. In contrast, both full-length A1 and A1ΔC protected EC from staurosporine (STS)-induced cell death, indicating that mitochondrial localization was not necessary for A1's cytoprotective function in human EC. In conclusion, our data uncover a regulatory role for the C-terminal domain of A1 in human EC: anchoring A1 to the mitochondrion, which conserves but is not necessary for its cytoprotective function, or by its absence freeing A1 from the mitochondrion and uncovering an additional anti-inflammatory effect.
Assuntos
Anti-Inflamatórios/metabolismo , Derme/metabolismo , Endotélio Vascular/metabolismo , Células Endoteliais da Veia Umbilical Humana/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Animais , Apoptose , Western Blotting , Bovinos , Proliferação de Células , Derme/citologia , Endotélio Vascular/citologia , Imunofluorescência , Regulação da Expressão Gênica , Células Endoteliais da Veia Umbilical Humana/citologia , Humanos , Molécula 1 de Adesão Intercelular/metabolismo , Luciferases/metabolismo , Antígenos de Histocompatibilidade Menor , NF-kappa B/genética , NF-kappa B/metabolismo , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas c-bcl-2/genéticaRESUMO
UNLABELLED: Liver regeneration is of major clinical importance in the setting of liver injury, resection, and transplantation. A20, a potent antiinflammatory and nuclear factor kappa B (NF-κB) inhibitory protein, has established pro-proliferative properties in hepatocytes, in part through decreasing expression of the cyclin dependent kinase inhibitor, p21. Both C-terminal (7-zinc fingers; 7Zn) and N-terminal (Nter) domains of A20 were required to decrease p21 and inhibit NF-κB. However, both independently increased hepatocyte proliferation, suggesting that additional mechanisms contributed to the pro-proliferative function of A20 in hepatocytes. We ascribed one of A20's pro-proliferative mechanisms to increased and sustained interleukin (IL)-6-induced signal transducer and activator of transcription 3 (STAT3) phosphorylation, as a result of decreased hepatocyte expression of the negative regulator of IL-6 signaling, suppressor of cytokine signaling 3 (SOCS3). This novel A20 function segregates with its 7Zn not Nter domain. Conversely, total and partial loss of A20 in hepatocytes increased SOCS3 expression, hampering IL-6-induced STAT3 phosphorylation. Following liver resection in mice pro-proliferative targets downstream of IL-6/STAT3 signaling were increased by A20 overexpression and decreased by A20 knockdown. In contrast, IL-6/STAT3 proinflammatory targets were increased in A20-deficient livers, and decreased or unchanged in A20 overexpressing livers. Upstream of SOCS3, levels of its microRNA regulator miR203 were significantly decreased in A20-deficient livers. CONCLUSION: A20 enhances IL-6/STAT3 pro-proliferative signals in hepatocytes by down-regulating SOCS3, likely through a miR203-dependent manner. This finding together with A20 reducing the levels of the potent cell cycle brake p21 establishes its pro-proliferative properties in hepatocytes and prompts the pursuit of A20-based therapies to promote liver regeneration and repair.
Assuntos
Proliferação de Células , Proteínas de Ligação a DNA/metabolismo , Interleucina-6/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Regeneração Hepática/fisiologia , Fígado/patologia , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais/fisiologia , Proteínas Supressoras da Sinalização de Citocina/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Células Cultivadas , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Cisteína Endopeptidases , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/genética , Hepatectomia , Hepatócitos/metabolismo , Hepatócitos/patologia , Peptídeos e Proteínas de Sinalização Intracelular/deficiência , Peptídeos e Proteínas de Sinalização Intracelular/genética , Fígado/metabolismo , Fígado/cirurgia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , MicroRNAs , Modelos Animais , NF-kappa B/metabolismo , Fosforilação , Proteína 3 Supressora da Sinalização de Citocinas , Proteína 3 Induzida por Fator de Necrose Tumoral alfa , Ubiquitina-Proteína Ligases/deficiência , Ubiquitina-Proteína Ligases/genéticaRESUMO
Inflammation induces the NF-κB dependent protein A20 in human renal proximal tubular epithelial cells (RPTEC), which secondarily contains inflammation by shutting down NF-κB activation. We surmised that inducing A20 without engaging the pro-inflammatory arm of NF-κB could improve outcomes in kidney disease. We showed that hepatocyte growth factor (HGF) increases A20 mRNA and protein levels in RPTEC without causing inflammation. Upregulation of A20 by HGF was NF-κB/RelA dependent as it was abolished by overexpressing IκBα or silencing p65/RelA. Unlike TNFα, HGF caused minimal IκBα and p65/RelA phosphorylation, with moderate IκBα degradation. Upstream, HGF led to robust and sustained AKT activation, which was required for p65 phosphorylation and A20 upregulation. While HGF treatment of RPTEC significantly increased A20 mRNA, it failed to induce NF-κB dependent, pro-inflammatory MCP-1, VCAM-1, and ICAM-1 mRNA. This indicates that HGF preferentially upregulates protective (A20) over pro-inflammatory NF-κB dependent genes. Upregulation of A20 supported the anti-inflammatory effects of HGF in RPTEC. HGF pretreatment significantly attenuated TNFα-mediated increase of ICAM-1, a finding partially reversed by silencing A20. In conclusion, this is the first demonstration that HGF activates an AKT-p65/RelA pathway to preferentially induce A20 but not inflammatory molecules. This could be highly desirable in acute and chronic renal injury where A20-based anti-inflammatory therapies are beneficial.
Assuntos
Fator de Crescimento de Hepatócito/farmacologia , Inflamação/prevenção & controle , Peptídeos e Proteínas de Sinalização Intracelular/genética , Túbulos Renais Proximais/efeitos dos fármacos , Túbulos Renais Proximais/metabolismo , NF-kappa B/metabolismo , Proteínas Nucleares/biossíntese , Proteínas Nucleares/genética , Células Cultivadas , Proteínas de Ligação a DNA , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Humanos , Inflamação/genética , Inflamação/metabolismo , Túbulos Renais Proximais/citologia , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transdução de Sinais/efeitos dos fármacos , Proteína 3 Induzida por Fator de Necrose Tumoral alfa , Regulação para Cima/efeitos dos fármacosRESUMO
The nuclear factor-kappaB inhibitory protein A20 demonstrates hepatoprotective abilities through combined antiapoptotic, anti-inflammatory, and pro-proliferative functions. Accordingly, overexpression of A20 in the liver protects mice from toxic hepatitis and lethal radical hepatectomy, whereas A20 knockout mice die prematurely from unfettered liver inflammation. The effect of A20 on oxidative liver damage, as seen in ischemia/reperfusion injury (IRI), is unknown. In this work, we evaluated the effects of A20 upon IRI using a mouse model of total hepatic ischemia. Hepatic overexpression of A20 was achieved by recombinant adenovirus (rAd.)-mediated gene transfer. Although only 10%-25% of control mice injected with saline or the control rAd.beta galactosidase survived IRI, the survival rate reached 67% in mice treated with rAd.A20. This significant survival advantage in rAd.A20-treated mice was associated with improved liver function, pathology, and repair potential. A20-treated mice had significantly lower bilirubin and aminotransferase levels, decreased hemorrhagic necrosis and steatosis, and increased hepatocyte proliferation. A20 protected against liver IRI by increasing hepatic expression of peroxisome proliferator-activated receptor alpha (PPARalpha), a regulator of lipid homeostasis and of oxidative damage. A20-mediated protection of hepatocytes from hypoxia/reoxygenation and H(2)O(2)-mediated necrosis was reverted by pretreatment with the PPARalpha inhibitor MK886. In conclusion, we demonstrate that PPARalpha is a novel target for A20 in hepatocytes, underscoring its novel protective effect against oxidative necrosis. By combining hepatocyte protection from necrosis and promotion of proliferation, A20-based therapies are well-poised to protect livers from IRI, especially in the context of small-for-size and steatotic liver grafts. Liver Transpl 15:1613-1621, 2009. (c) 2009 AASLD.
Assuntos
Doença Hepática Induzida por Substâncias e Drogas/fisiopatologia , Cisteína Endopeptidases/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Transplante de Fígado , PPAR alfa/metabolismo , Traumatismo por Reperfusão/fisiopatologia , Animais , Divisão Celular/fisiologia , Células Cultivadas , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Doença Hepática Induzida por Substâncias e Drogas/patologia , Cisteína Endopeptidases/genética , Fígado Gorduroso/metabolismo , Fígado Gorduroso/patologia , Fígado Gorduroso/fisiopatologia , Galactosamina/toxicidade , Expressão Gênica/fisiologia , Hepatectomia , Hepatócitos/citologia , Hepatócitos/fisiologia , Peróxido de Hidrogênio/toxicidade , Peptídeos e Proteínas de Sinalização Intracelular/genética , Lipopolissacarídeos/toxicidade , Falência Hepática Aguda/metabolismo , Falência Hepática Aguda/patologia , Falência Hepática Aguda/fisiopatologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Necrose , Oxidantes/toxicidade , PPAR alfa/genética , Traumatismo por Reperfusão/metabolismo , Traumatismo por Reperfusão/patologia , Proteína 3 Induzida por Fator de Necrose Tumoral alfaRESUMO
BACKGROUND: Inflammation is central to the pathogenesis of transplant arteriosclerosis (TA). We questioned whether physiologic levels of anti-inflammatory A20 influence TA severity. METHODS: We performed major histocompatibility complex mismatched aorta to carotid artery interposition grafts, using wild type (WT) or A20 heterozygote (HET) C57BL/6 (H-2) donors and BALB/c (H-2) recipients, and conversely BALB/c donors and WT/HET recipients. We analyzed aortic allografts by histology, immunohistochemistry, immunofluorescence, and gene profiling (quantitative real-time reverse-transcriptase polymerase chain reaction). We validated select in vivo A20 targets in human and mouse smooth muscle cell (SMC) cultures. RESULTS: We noted significantly greater intimal hyperplasia in HET versus WT allografts, indicating aggravated TA. Inadequate upregulation of A20 in HET allografts after transplantation was associated with excessive NF-кB activation, gauged by higher levels of IkBα, p65, VCAM-1, ICAM-1, CXCL10, CCL2, TNF, and IL-6 (mostly localized to SMC). Correspondingly, cytokine-induced upregulation of TNF and IL-6 in human and mouse SMC cultures inversely correlated with A20 expression. Aggravated TA in HET versus WT allografts correlated with increased intimal SMC proliferation, and a higher number of infiltrating IFNγ and Granzyme B CD4 T cells and natural killer cells, and lower number of FoxP3 regulatory T cells. A20 haploinsufficiency in allograft recipients did not influence TA. CONCLUSIONS: A20 haploinsufficiency in vascular allografts aggravates lesions of TA by exacerbating inflammation, SMC proliferation, and infiltration of pathogenic T cells. A20 single nucleotide polymorphisms associating with lower A20 expression or function in donors of vascularized allografts may inform risk and severity of TA, highlighting the clinical implications of our findings.
Assuntos
Aorta/transplante , Arteriosclerose/etiologia , Haploinsuficiência , Complicações Pós-Operatórias/etiologia , Proteína 3 Induzida por Fator de Necrose Tumoral alfa/genética , Animais , Arteriosclerose/genética , Arteriosclerose/imunologia , Humanos , Interferon gama/biossíntese , Interleucina-6/biossíntese , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Miócitos de Músculo Liso/fisiologia , Complicações Pós-Operatórias/genética , Complicações Pós-Operatórias/imunologia , Transplante Homólogo , Fator de Necrose Tumoral alfa/biossíntese , Túnica Íntima/patologiaRESUMO
L-2-Hydroxyglutaric (LGA) and D-2-hydroxyglutaric (DGA) acids are the characteristic metabolites accumulating in the neurometabolic disorders known as L-2-hydroxyglutaric aciduria and D-2-hydroxyglutaric aciduria, respectively. Although these disorders are predominantly characterized by severe neurological symptoms, the neurotoxic mechanisms of brain damage are virtually unknown. In this study we have evaluated the role of LGA and DGA at concentrations ranging from 0.01 to 5.0 mM on various parameters of energy metabolism in cerebral cortex slices and homogenates of 30-day-old Wistar rats, namely glucose uptake, CO(2) production and the respiratory chain enzyme activities of complexes I to IV. DGA significantly decreased glucose utilization (2.5 and 5.0 mM) by brain homogenates and CO(2) production (5 mM) by brain homogenates and slices, whereas LGA had no effect on either measurement. Furthermore, DGA significantly inhibited cytochrome c oxidase activity (complex IV) (EC 1.9.3.1) in a dose-dependent manner (35-95%) at doses as low as 0.5 mM, without compromising the other respiratory chain enzyme activities. In contrast, LGA did not interfere with these activities. Our results suggest that the strong inhibition of cytochrome c oxidase activity by increased levels of DGA could be related to the neurodegeneration of patients affected by D-2-hydroxyglutaric aciduria.
Assuntos
Córtex Cerebral/enzimologia , Complexo IV da Cadeia de Transporte de Elétrons/antagonistas & inibidores , Glutaratos/farmacologia , Músculo Esquelético/enzimologia , Animais , Dióxido de Carbono/metabolismo , Córtex Cerebral/efeitos dos fármacos , Relação Dose-Resposta a Droga , Complexo III da Cadeia de Transporte de Elétrons/antagonistas & inibidores , Metabolismo Energético , Glucose/metabolismo , Humanos , Técnicas In Vitro , Cinética , Músculo Esquelético/efeitos dos fármacos , Ratos , EstereoisomerismoRESUMO
D-2-Hydroxyglutaric acid (DGA) is the biochemical hallmark of patients affected by the neurometabolic disorder known as D-2-hydroxyglutaric aciduria (DHGA). Although this disease is predominantly characterized by severe neurological findings, the underlying mechanisms of brain injury are virtually unknown. In the present study, we investigated the effect of DGA on total, cytosolic, and mitochondrial creatine kinase (CK) activities from cerebral cortex of 30-day-old Wistar rats. Total CK activity (tCK) was measured in whole cell homogenates, whereas cytosolic and mitochondrial activities were measured in the cytosolic and mitochondrial preparations from cerebral cortex. We verified that CK activities were significantly inhibited by DGA (11-34% inhibition) at concentrations as low as 0.25 mM, being the mitochondrial fraction the most affected activity. Kinetic studies revealed that the inhibitory effect of DGA was non-competitive in relation to phosphocreatine. We also observed that this inhibition was fully prevented by pre-incubation of the homogenates with reduced glutathione, suggesting that the inhibitory effect of DGA on tCK activity is possibly mediated by oxidation of essential thiol groups of the enzyme. Considering the importance of CK activity for brain metabolism homeostasis, our results suggest that inhibition of this enzyme by increased levels of DGA may be related to the neurodegeneration of patients affected by DHGA.
Assuntos
Córtex Cerebral/enzimologia , Creatina Quinase/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Glutaratos/farmacologia , Animais , Ácido Ascórbico/farmacologia , Córtex Cerebral/efeitos dos fármacos , Creatina Quinase/metabolismo , Citosol/efeitos dos fármacos , Citosol/enzimologia , Sequestradores de Radicais Livres/farmacologia , Glutationa/farmacologia , Técnicas In Vitro , Cinética , Masculino , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/enzimologia , NG-Nitroarginina Metil Éster/farmacologia , Óxido Nítrico Sintase/antagonistas & inibidores , Oxirredução , Ratos , Ratos Wistar , Vitamina E/farmacologiaRESUMO
L-2-Hydroxyglutaric acid (LGA) is the biochemical hallmark of patients affected by the neurometabolic disorder known as L-2-hydroxyglutaric aciduria (LHGA). Although this disorder is predominantly characterized by severe neurological findings and pronounced cerebellum atrophy, the neurotoxic mechanisms of brain injury are virtually unknown. In the present study, we investigated the effect of LGA, at 0.25-5mM concentrations, on total creatine kinase (tCK) activity from cerebellum, cerebral cortex, cardiac muscle and skeletal muscle homogenates of 30-day-old Wistar rats. CK activity was measured also in the cytosolic (Cy-CK) and mitochondrial (Mi-CK) fractions from cerebellum. We verified that tCK activity was significantly inhibited by LGA in the cerebellum, but not in cerebral cortex, cardiac muscle and skeletal muscle. Furthermore, CK activity from the mitochondrial fraction was inhibited by LGA, whereas that from the cytosolic fraction of cerebellum was not affected by the acid. Kinetic studies revealed that the inhibitory effect of LGA on Mi-CK was non-competitive in relation to phosphocreatine. Finally, we verified that the inhibitory effect of LGA on tCK was fully prevented by pre-incubation of the homogenates with reduced glutathione (GSH), suggesting that this inhibition is possibly mediated by oxidation of essential thiol groups of the enzyme. Considering the importance of creatine kinase activity for energy homeostasis, our results suggest that the selective inhibition of this enzyme activity by increased levels of LGA could be possibly related to the cerebellar degeneration characteristically found in patients affected by L-2-hydroxyglutaric aciduria.
Assuntos
Cerebelo/embriologia , Cerebelo/enzimologia , Creatina Quinase/antagonistas & inibidores , Creatina Quinase/metabolismo , Glutaratos/farmacologia , Isoenzimas/antagonistas & inibidores , Isoenzimas/metabolismo , Animais , Ácido Ascórbico/farmacologia , Encefalopatias Metabólicas Congênitas/metabolismo , Cerebelo/efeitos dos fármacos , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/embriologia , Córtex Cerebral/enzimologia , Creatina Quinase Mitocondrial , Glutationa/farmacologia , Coração/efeitos dos fármacos , Coração/embriologia , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/enzimologia , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/embriologia , Músculo Esquelético/enzimologia , Miocárdio/enzimologia , NG-Nitroarginina Metil Éster/farmacologia , Especificidade de Órgãos , Ratos , Ratos Wistar , alfa-Tocoferol/farmacologiaRESUMO
Interactions between neural progenitor cells (NPC) and endothelial cells (EC) from adult vascular beds have been well explored previously. However, the factors and signaling mechanisms that regulate neurogenesis and angiogenesis are most prevalent during embryonic development. This study aimed to determine whether embryonic brain endothelial cells from the periventricular region (PVEC) present an advantage over adult brain EC in supporting NPC growth and differentiation. PVEC were isolated from E15 mouse brains, processed, and sorted with immunomagnetic beads using antibodies against CD31/PECAM. On immunofluorescence (IF) staining, nearly all cells were positive for EC markers CD31 and CD144/VE-Cadherin. In proliferation studies, NPC proliferation was highest in transwell co-culture with PVEC, approximately 2.3 fold increase compared to baseline versus 1.4 fold increase when co-cultured with adult brain endothelial cells (ABEC). These results correlated with the PVEC mediated delay in NPC differentiation, evidenced by high expression of progenitor marker Nestin evaluated by IF staining. Upon further characterization of PVEC in an angiogenesis assay measuring cord length, PVEC exhibited a high capacity to form cords in basal conditions compared to ABEC. This was enhanced in the presence of NPC, with both cell types displaying a preferential structural alignment resembling neurovascular networks. PVEC also expressed high Vegfa levels at baseline in comparison to NPC and ABEC. Vegfa levels increased when co-cultured with NPC. We demonstrate that PVEC and NPC co-cultures act synergistically to promote the formation of a neurovascular unit through dynamic and reciprocal communication. Our results suggest that PVEC/NPC could provide promising neuro-regenerative therapies for patients suffering brain injuries.
Assuntos
Ventrículos Cerebrais/embriologia , Células Endoteliais/citologia , Neovascularização Fisiológica/fisiologia , Células-Tronco Neurais/citologia , Animais , Células Cultivadas , Células Endoteliais/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Células-Tronco Neurais/metabolismoRESUMO
BACKGROUND: Transplant arteriosclerosis (TA) is the pathognomonic feature of chronic rejection, the primary cause of allograft failure. We have shown that the NF-κB inhibitory protein A20 exerts vasculoprotective effects in endothelial and smooth muscle cells (SMC), and hence is a candidate to prevent TA. We sought direct proof for this hypothesis. METHODS: Fully mismatched, C57BL/6 (H2) into BALB/c (H2), aorta to carotid allografts were preperfused with saline, recombinant A20 adenovirus (rAd.A20) or rAd.ß-galactosidase (ß-gal), implanted, harvested 4 weeks after transplantation, and analyzed by histology, immunohistochemistry, and immunofluorescence staining. We measured indoleamine 2,3-dioxygenase, interleukin-6, and transforming growth factor-ß mRNA and protein levels in nontransduced, and rAd.A20 or rAd.ß-gal-transduced human SMC cultures after cytokine treatment. RESULTS: Vascular overexpression of A20 significantly reduced TA lesions. This correlated with decreased graft inflammation and increased apoptosis of neointimal SMC. Paradoxically, T-cell infiltrates increased in A20-expressing allografts, including the immunoprivileged media, which related to A20 preventing indoleamine 2,3-dioxygenase upregulation in SMC. However, infiltrating T cells were predominantly T-regulatory cells (CD25+/Forkhead Box P3 [FoxP3+]). This agrees with A20 inhibiting interleukin-6 and promoting transforming growth factor-ß production by medial SMC and in SMC cultures exposed to cytokines, which favors differentiation of regulatory over pathogenic T cells. CONCLUSIONS: In summary, A20 prevents immune-mediated remodeling of vascular allografts, therefore reduces TA lesions by affecting apoptotic and inflammatory signals and modifying the local cytokine milieu to promote an immunoregulatory response within the vessel wall. This highlights a novel function for A20 in local immunosurveillance, which added to its vasculoprotective effects, supports its therapeutic promise in TA.
Assuntos
Aorta/transplante , Arteriosclerose/imunologia , Rejeição de Enxerto/imunologia , Imunidade Inata/imunologia , Inflamação/imunologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Adenoviridae/genética , Animais , Aorta/metabolismo , Aorta/patologia , Apoptose , Arteriosclerose/complicações , Arteriosclerose/metabolismo , Artérias Carótidas/metabolismo , Artérias Carótidas/patologia , Artérias Carótidas/cirurgia , Células Cultivadas , Cisteína Endopeptidases/genética , Cisteína Endopeptidases/metabolismo , Citocinas/metabolismo , Rejeição de Enxerto/etiologia , Rejeição de Enxerto/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Modelos Animais , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patologia , Transplante Homólogo , Proteína 3 Induzida por Fator de Necrose Tumoral alfaRESUMO
BACKGROUND: Liver regeneration is clinically of major importance in the setting of liver injury, resection or transplantation. We have demonstrated that the NF-κB inhibitory protein A20 significantly improves recovery of liver function and mass following extended liver resection (LR) in mice. In this study, we explored the Systems Biology modulated by A20 following extended LR in mice. METHODOLOGY AND PRINCIPAL FINDINGS: We performed transcriptional profiling using Affymetrix-Mouse 430.2 arrays on liver mRNA retrieved from recombinant adenovirus A20 (rAd.A20) and rAd.ßgalactosidase treated livers, before and 24 hours after 78% LR. A20 overexpression impacted 1595 genes that were enriched for biological processes related to inflammatory and immune responses, cellular proliferation, energy production, oxidoreductase activity, and lipid and fatty acid metabolism. These pathways were modulated by A20 in a manner that favored decreased inflammation, heightened proliferation, and optimized metabolic control and energy production. Promoter analysis identified several transcriptional factors that implemented the effects of A20, including NF-κB, CEBPA, OCT-1, OCT-4 and EGR1. Interactive scale-free network analysis captured the key genes that delivered the specific functions of A20. Most of these genes were affected at basal level and after resection. We validated a number of A20's target genes by real-time PCR, including p21, the mitochondrial solute carriers SLC25a10 and SLC25a13, and the fatty acid metabolism regulator, peroxisome proliferator activated receptor alpha. This resulted in greater energy production in A20-expressing livers following LR, as demonstrated by increased enzymatic activity of cytochrome c oxidase, or mitochondrial complex IV. CONCLUSION: This Systems Biology-based analysis unravels novel mechanisms supporting the pro-regenerative function of A20 in the liver, by optimizing energy production through improved lipid/fatty acid metabolism, and down-regulated inflammation. These findings support pursuit of A20-based therapies to improve patients' outcomes in the context of extreme liver injury and extensive LR for tumor treatment or donation.
Assuntos
Metabolismo Energético , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Metabolismo dos Lipídeos , Regeneração Hepática/fisiologia , Proteínas Nucleares/metabolismo , Animais , Sítios de Ligação , Proliferação de Células , Proteínas de Ligação a DNA , Regulação da Expressão Gênica , Redes Reguladoras de Genes , Hepatócitos/metabolismo , Humanos , Inflamação/metabolismo , Inflamação/patologia , Fígado/metabolismo , Fígado/patologia , Fígado/cirurgia , Camundongos , Regiões Promotoras Genéticas/genética , Reprodutibilidade dos Testes , Fatores de Transcrição/metabolismo , Transcrição Gênica , Proteína 3 Induzida por Fator de Necrose Tumoral alfaRESUMO
OBJECTIVE: Neointimal hyperplasia is an inflammatory and proliferative process that occurs as a result of injury to the vessel wall. We have shown that the homeostatic protein A20 prevents neointimal hyperplasia by affecting endothelial cell (EC) and smooth muscle cell (SMC) responses to injury. In this work, we questioned whether A20 impacts other pathogenic effectors of neointimal hyperplasia including homing of monocyte/macrophages and EC/SMC precursors to the site of vascular injury, vascular endothelial growth factor (VEGF) secretion, and adventitial neovascularization. METHODS AND RESULTS: Carotid balloon angioplasty was performed on rat recipients of a bone marrow transplant from green fluorescent rats. Adenoviral delivery of A20 prevented neointimal hyperplasia and decreased macrophage infiltration. This was associated with decreased ICAM-1 and MCP-1 expression in vitro. Additionally, A20 reduced neovascularization in the adventitia of balloon injured carotid arteries, which correlated with fewer VEGF positive cells. CONCLUSIONS: A20 downregulates adhesion markers, chemokine production, and adventitial angiogenesis, all of which are required for macrophage trafficking to sites of vascular injury. This, in turn, diminishes the inflammatory milieu to prevent neointimal hyperplasia.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Macrófagos/citologia , Proteínas Nucleares/metabolismo , Animais , Células da Medula Óssea/citologia , Movimento Celular , Regulação para Baixo , Endotélio Vascular/patologia , Humanos , Hiperplasia/patologia , Macrófagos/metabolismo , Masculino , Monócitos/metabolismo , Neointima/patologia , Neovascularização Patológica , Ratos , Ratos Sprague-Dawley , Proteína 3 Induzida por Fator de Necrose Tumoral alfa , Células U937 , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
BACKGROUND: Accelerated atherosclerosis is the leading cause of morbidity and mortality in diabetic patients. Hyperglycemia is a recognized independent risk factor for heightened atherogenesis in diabetes mellitus (DM). However, our understanding of the mechanisms underlying glucose damage to the vasculature remains incomplete. METHODOLOGY/PRINCIPAL FINDINGS: High glucose and hyperglycemia reduced upregulation of the NF-κB inhibitory and atheroprotective protein A20 in human coronary endothelial (EC) and smooth muscle cell (SMC) cultures challenged with Tumor Necrosis Factor alpha (TNF), aortae of diabetic mice following Lipopolysaccharide (LPS) injection used as an inflammatory insult and in failed vein-grafts of diabetic patients. Decreased vascular expression of A20 did not relate to defective transcription, as A20 mRNA levels were similar or even higher in EC/SMC cultured in high glucose, in vessels of diabetic C57BL/6 and FBV/N mice, and in failed vein grafts of diabetic patients, when compared to controls. Rather, decreased A20 expression correlated with post-translational O-Glucosamine-N-Acetylation (O-GlcNAcylation) and ubiquitination of A20, targeting it for proteasomal degradation. Restoring A20 levels by inhibiting O-GlcNAcylation, blocking proteasome activity, or overexpressing A20, blocked upregulation of the receptor for advanced glycation end-products (RAGE) and phosphorylation of PKCßII, two prime atherogenic signals triggered by high glucose in EC/SMC. A20 gene transfer to the aortic arch of diabetic ApoE null mice that develop accelerated atherosclerosis, attenuated vascular expression of RAGE and phospho-PKCßII, significantly reducing atherosclerosis. CONCLUSIONS: High glucose/hyperglycemia regulate vascular A20 expression via O-GlcNAcylation-dependent ubiquitination and proteasomal degradation. This could be key to the pathogenesis of accelerated atherosclerosis in diabetes.
Assuntos
Apolipoproteínas E/genética , Aterosclerose/metabolismo , Cisteína Endopeptidases/genética , Diabetes Mellitus Experimental/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Ubiquitina/química , Animais , Cisteína Endopeptidases/metabolismo , Glicosilação , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Lipopolissacarídeos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Miócitos de Músculo Liso/citologia , NF-kappa B/metabolismo , Receptor para Produtos Finais de Glicação Avançada , Receptores Imunológicos/metabolismo , Proteína 3 Induzida por Fator de Necrose Tumoral alfaRESUMO
D-2-Hydroxyglutaric aciduria (DHGA) is a neurometabolic disorder biochemically characterized by tissue accumulation and excretion of high amounts of D-2-hydroxyglutaric acid (DGA). Although the affected patients have predominantly severe neurological findings, the underlying mechanisms of brain injury are virtually unknown. In previous studies we have demonstrated that DGA, at concentrations as low as 0.25 mM, significantly decreased creatine kinase activity and other parameters of energy metabolism in cerebral cortex of young rats. In the present study, we investigated the effect of DGA (0.25-5 mM) on total creatine kinase (tCK) activity, as well as on CK activity in cytosolic (Cy-CK) and mitochondrial (Mi-CK) preparations from cerebellum of 30-day-old Wistar rats in order to test whether the inhibitory effect of DGA on CK was tissue specific. We verified that tCK (22% inhibition) and Mi-CK (40% inhibition) activities were moderately inhibited by DGA at concentrations of 2.5 mM and higher, in contrast to Cy-CK, which was not affected by the acid. Kinetic studies revealed that the inhibitory effect of DGA was noncompetitive in relation to phosphocreatine. We also observed that this inhibition was fully prevented by preincubation of the homogenates with reduced glutathione, suggesting that the inhibition of CK activity by DGA is possibly mediated by modification of essential thiol groups of the enzyme. Our present results therefore demonstrate a relatively weak inhibitory effect of DGA on cerebellum Mi-CK activity, as compared to that provoked in cerebral cortex, and may possibly be related to the neuropathology of DHGA, characterized by cerebral cortex abnormalities.