RESUMEN
Cerebral cavernous malformations (CCMs) are vascular lesions predominantly developing in the central nervous system (CNS), with no effective treatments other than surgery. Loss-of-function mutation in CCM1/krev interaction trapped 1 (KRIT1), CCM2, or CCM3/programmed cell death 10 (PDCD10) causes lesions that are characterized by abnormal vascular integrity. Vascular endothelial cadherin (VE-cadherin), a major regulator of endothelial cell (EC) junctional integrity is strongly disorganized in ECs lining the CCM lesions. We report here that microRNA-27a (miR-27a), a negative regulator of VE-cadherin, is elevated in ECs isolated from mouse brains developing early CCM lesions and in cultured ECs with CCM1 or CCM2 depletion. Furthermore, we show miR-27a acts downstream of kruppel-like factor (KLF)2 and KLF4, two known key transcription factors involved in CCM lesion development. Using CD5-2 (a target site blocker [TSB]) to prevent the miR-27a/VE-cadherin mRNA interaction, we present a potential therapy to increase VE-cadherin expression and thus rescue the abnormal vascular integrity. In CCM1- or CCM2-depleted ECs, CD5-2 reduces monolayer permeability, and in Ccm1 heterozygous mice, it restores dermal vessel barrier function. In a neonatal mouse model of CCM disease, CD5-2 normalizes vasculature and reduces vascular leakage in the lesions, inhibits the development of large lesions, and significantly reduces the size of established lesions in the hindbrain. Furthermore, CD5-2 limits the accumulation of inflammatory cells in the lesion area. Our work has established that VE-cadherin is a potential therapeutic target for normalization of the vasculature and highlights that targeting miR-27a/VE-cadherin interaction by CD5-2 is a potential novel therapy for the devastating disease, CCM.
Asunto(s)
Antígenos CD/metabolismo , Cadherinas/metabolismo , Hemangioma Cavernoso del Sistema Nervioso Central/genética , MicroARNs/metabolismo , Animales , Regulación hacia Abajo/genética , Hemangioma Cavernoso del Sistema Nervioso Central/patología , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Factor 4 Similar a Kruppel , Factores de Transcripción de Tipo Kruppel/metabolismo , Masculino , Ratones Endogámicos C57BL , MicroARNs/genética , Rombencéfalo/irrigación sanguínea , Rombencéfalo/patología , Regulación hacia Arriba/genética , Proteína de Unión al GTP rhoA/metabolismoRESUMEN
MiR-181 expression levels increased in hepatocellular carcinoma (HCC) compared to non-cancerous tissues. MiR-181 has been widely reported as a possible driver of tumourigenesis but also acts as a tumour suppressor. In addition, the miR-181 family regulates the development and function of immune and vascular cells, which play vital roles in the progression of tumours. More complicatedly, many genes have been identified as miR-181 targets to mediate the effects of miR-181. However, the role of miR-181 in the development of primary tumours remains largely unexplored. We aimed to examine the function of miR-181 and its vital mediators in the progression of diethylnitrosamine-induced primary liver cancers in mice. The size of liver tumours was significantly reduced by 90% in global (GKO) or liver-specific (LKO) 181ab1 knockout mice but not in hematopoietic and endothelial lineage-specific knockout mice, compared to WT mice. In addition, the number of tumours was significantly reduced by 50% in GKO mice. Whole-genome RNA-seq analysis and immunohistochemistry showed that epithelial-mesenchymal transition was partially reversed in GKO tumours compared to WT tumours. The expression of CBX7, a confirmed miR-181 target, was up-regulated in GKO compared to WT tumours. Stable CBX7 expression was achieved with an AAV/Transposase Hybrid-Vector System and up-regulated CBX7 expression inhibited liver tumour progression in WT mice. Hepatic CBX7 deletion restored the progression of LKO liver tumours. MiR-181a expression was the lowest and CBX7 expression the highest in iClust2 and 3 subclasses of human HCC compared to iClust1. Gene expression profiles of GKO tumours overlapped with low-proliferative peri-portal-type HCCs. Liver-specific loss of miR-181ab1 inhibited primary liver tumour progression via up-regulating CBX7 expression, but tumour induction requires both hepatic and non-hepatic miR-181. Also, miR-181ab1-deficient liver tumours may resemble low-proliferative periportal-type human HCC. miR-181 was increased with liver tumour growth. More miR-181, darker colour and higher shape. CBX7 was very low in pericentral hepatocytes, increased in early liver tumours, but reduced in advanced liver tumours. Its levels were maintained in miR-181 KO liver tumours. In tumours (T), brown (darker is more) represents miR-181, the blue circle (thicker is more) represents CBX7.
Asunto(s)
Carcinoma Hepatocelular , Neoplasias Hepáticas , MicroARNs , Animales , Carcinoma Hepatocelular/inducido químicamente , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Ratones , MicroARNs/genética , MicroARNs/metabolismo , Complejo Represivo Polycomb 1/genética , Complejo Represivo Polycomb 1/metabolismo , Regulación hacia Arriba/genéticaRESUMEN
Sphingolipid dysregulation is often associated with insulin resistance, while the enzymes controlling sphingolipid metabolism are emerging as therapeutic targets for improving insulin sensitivity. We report herein that sphingosine kinase 2 (SphK2), a key enzyme in sphingolipid catabolism, plays a critical role in the regulation of hepatic insulin signaling and glucose homeostasis both in vitro and in vivo. Hepatocyte-specific Sphk2 knockout mice exhibit pronounced insulin resistance and glucose intolerance. Likewise, SphK2-deficient hepatocytes are resistant to insulin-induced activation of the phosphoinositide 3-kinase (PI3K)-Akt-FoxO1 pathway and elevated hepatic glucose production. Mechanistically, SphK2 deficiency leads to the accumulation of sphingosine that, in turn, suppresses hepatic insulin signaling by inhibiting PI3K activation in hepatocytes. Either reexpressing functional SphK2 or pharmacologically inhibiting sphingosine production restores insulin sensitivity in SphK2-deficient hepatocytes. In conclusion, the current study provides both experimental findings and mechanistic data showing that SphK2 and sphingosine in the liver are critical regulators of insulin sensitivity and glucose homeostasis.
Asunto(s)
Glucosa/metabolismo , Insulina/metabolismo , Hígado/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Animales , Línea Celular Tumoral , Femenino , Hepatocitos/enzimología , Hepatocitos/metabolismo , Homeostasis , Humanos , Hígado/enzimología , Masculino , Ratones , Ratones Noqueados , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Esfingolípidos/metabolismoRESUMEN
BACKGROUND: Vascular endothelial cell alignment in the direction of flow is an adaptive response that protects against aortic diseases such as atherosclerosis. The RhoGTPases are known to regulate this alignment. We have shown previously that ARHGAP18 in endothelial cells is a negative regulator of RhoC and its expression is essential in flow-mediated alignment. Depletion of ARHGAP18 inhibits alignment and results in the induction of a pro-inflammatory phenotype. In embryogenesis, ARHGAP18 was identified as a downstream effector of the Yes-associated protein, YAP, which regulates cell shape and size. METHODS: We have used siRNA technology to deplete either ARHGAP18 or YAP in human endothelial cells. The in vitro studies were performed under athero-protective, laminar flow conditions. The analysis of YAP activity was also investigated, using high performance confocal imaging, in our ARHGAP18 knockout mutant mice. RESULTS: We show here that loss of ARHGAP18, although decreasing the expression of YAP results in its nuclear localisation consistent with activation. We further show that depletion of YAP itself results in its activation as defined by an in increase in its nuclear localisation and an increase in the YAP target gene, CyR61. Depletion of YAP, similar to that observed for ARHGAP18 depletion, results in loss of endothelial cell alignment under high shear stress mediated flow and also in the activation of NFkB, as determined by p65 nuclear localisation. In contrast, ARHGAP18 overexpression results in upregulation of YAP, its phosphorylation, and a decrease in the YAP target gene Cyr61, consistent with YAP inactivation. Finally, in ARHGAP18 deleted mice, in regions where there is a loss of endothelial cell alignment, a situation associated with a priming of the cells to a pro-inflammatory phenotype, YAP shows nuclear localisation. CONCLUSION: Our results show that YAP is downstream of ARHGAP18 in mature endothelial cells and that this pathway is involved in the athero-protective alignment of endothelial cells under laminar shear stress. ARHGAP18 depletion leads to a disruption of the junctions as seen by loss of VE-Cadherin localisation to these regions and a concomitant localisation of YAP to the nucleus.
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Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Activadoras de GTPasa/metabolismo , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Reología , Factores de Transcripción/metabolismo , Proteína rhoC de Unión a GTP/metabolismo , Animales , Aorta/metabolismo , Proteínas Activadoras de GTPasa/deficiencia , Eliminación de Gen , Humanos , Masculino , Ratones Noqueados , Proteínas Señalizadoras YAPRESUMEN
AIMS/HYPOTHESIS: A major feature of diabetic retinopathy is breakdown of the blood-retinal barrier, resulting in macular oedema. We have developed a novel oligonucleotide-based drug, CD5-2, that specifically increases expression of the key junctional protein involved in barrier integrity in endothelial cells, vascular-endothelial-specific cadherin (VE-cadherin). CD5-2 prevents the mRNA silencing by the pro-angiogenic microRNA, miR-27a. CD5-2 was evaluated in animal models of ocular neovascularisation and vascular leak to determine its potential efficacy for diabetic retinopathy. METHODS: CD5-2 was tested in three mouse models of retinal dysfunction: conditional Müller cell depletion, streptozotocin-induced diabetes and oxygen-induced retinopathy. Vascular permeability in the Müller cell-knockout model was assessed by fluorescein angiography. The Evans Blue leakage method was used to determine vascular permeability in streptozotocin- and oxygen-induced retinopathy models. The effects of CD5-2 on retinal neovascularisation, inter-endothelial junctions and pericyte coverage in streptozotocin- and oxygen-induced retinopathy models were determined by staining for isolectin-B4, VE-cadherin and neural/glial antigen 2 (NG2). Blockmir CD5-2 localisation in diseased retina was determined using fluorescent in situ hybridisation. The effects of CD5-2 on VE-cadherin expression and in diabetic retinopathy-associated pathways, such as the transforming growth factor beta (TGF-ß) and wingless/integrated (WNT) pathway, were confirmed using western blot of lysates from HUVECs, a mouse brain endothelial cell line and a VE-cadherin null mouse endothelial cell line. RESULTS: CD5-2 penetrated the vasculature of the eye in the oxygen-induced retinopathy model. Treatment of diseased mice with CD5-2 resulted in reduced vascular leak in all three animal models, enhanced expression of VE-cadherin in the microvessels of the eye and improved pericyte coverage of the retinal vasculature in streptozotocin-induced diabetic models and oxygen-induced retinopathy models. Further, CD5-2 reduced the activation of retinal microglial cells in the streptozotocin-induced diabetic model. The positive effects of CD5-2 seen in vivo were further confirmed in vitro by increased protein expression of VE-cadherin, SMAD2/3 activity, and platelet-derived growth factor B (PDGF-B). CONCLUSIONS/INTERPRETATION: CD5-2 has therapeutic potential for individuals with vascular-leak-associated retinal diseases based on its ease of delivery and its ability to reverse vascular dysfunction and inflammatory aspects in three animal models of retinopathy.
Asunto(s)
Antígenos CD/metabolismo , Cadherinas/metabolismo , Diabetes Mellitus Experimental/tratamiento farmacológico , Retinopatía Diabética/tratamiento farmacológico , Oligonucleótidos/uso terapéutico , Animales , Barrera Hematorretinal/metabolismo , Permeabilidad Capilar , Diabetes Mellitus Experimental/metabolismo , Retinopatía Diabética/metabolismo , Ratones , Retina/metabolismo , Vasos Retinianos/metabolismoRESUMEN
RATIONALE: Thoracic aortic aneurysm (TAA) is a potentially lethal condition, which can affect individuals of all ages. TAA may be complicated by the sudden onset of life-threatening dissection or rupture. The underlying mechanisms leading to TAA formation, particularly in the nonsyndromal idiopathic group of patients, are not well understood. Thus, identification of new genes and targets that are involved in TAA pathogenesis are required to help prevent and reverse the disease phenotype. OBJECTIVE: Here we explore the role of ARHGAP18, a novel Rho GAP expressed by smooth muscle cells (SMCs), in the pathogenesis of TAA. METHODS AND RESULTS: Using human and mouse aortic samples, we report that ARHGAP18 levels were significantly reduced in the SMC layer of aortic aneurysms. Arhgap18 global knockout (Arhgap18-/-) mice exhibited a highly synthetic, proteolytic, and proinflammatory smooth muscle phenotype under basal conditions and when challenged with angiotensin II, developed TAA with increased frequency and severity compared with littermate controls. Chromatin immunoprecipitation studies revealed this phenotype is partly associated with strong enrichment of H3K4me3 and depletion of H3K27me3 at the MMP2 and TNF-α promoters in Arhgap18-deficient SMC. We further show that TAA formation in the Arhgap18-/- mice is associated with loss of Akt activation. The abnormal SMC phenotype observed in the Arhgap18-/- mice can be partially rescued by pharmacological treatment with the mTORC1 inhibitor rapamycin, which reduces the synthetic and proinflammatory phenotype of Arhgap18-deficient SMC. CONCLUSION: We have identified ARHGAP18 as a novel protective gene against TAA formation and define an additional target for the future development of treatments to limit TAA pathogenesis.
Asunto(s)
Aneurisma de la Aorta Torácica/metabolismo , Aneurisma de la Aorta Torácica/prevención & control , Proteínas Activadoras de GTPasa/deficiencia , Mediadores de Inflamación/metabolismo , Músculo Liso Vascular/metabolismo , Miocitos del Músculo Liso/metabolismo , Animales , Aneurisma de la Aorta Torácica/genética , Proteínas Activadoras de GTPasa/genética , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , FenotipoRESUMEN
Sphingolipid metabolites have emerged playing important roles in the pathogenesis of nonalcoholic fatty liver disease, whereas the underlying mechanism remains largely unknown. In the present study, we provide both in vitro and in vivo evidence showing a pathogenic role of sphingosine kinase 1 (SphK1) in hepatocellular steatosis. We found that levels of SphK1 expression were significantly increased in steatotic hepatocytes. Enforced overexpression of SphK1 or treatment with sphingosine 1-phosphate (S1P) markedly enhanced hepatic lipid accumulation. In contrast, the siRNA-mediated knockdown of SphK1 or S1P receptors, S1P2 and S1P3, profoundly inhibited lipid accumulation in hepatocytes. Moreover, Sphk1(-/-) mice exhibited a significant amelioration of hepatosteatosis under diet-induced obese (DIO) conditions, compared to wild-type littermates. In addition, DIO-induced up-regulation of PPARγ and its target genes were significantly reduced by SphK1 deficiency. Furthermore, treatment of hepatocytes with S1P induces a dose-dependent increase in PPARγ expression at the transcriptional level. Blockage of S1P receptors and the Akt-mTOR signaling profoundly inhibited S1P-induced PPARγ expression. Notably, down-regulation of PPARγ by using its siRNA significantly diminished the pro-steatotic effect of SphK1/S1P. Thus, the study demonstrates a new pathway connecting SphK1 and PPARγ involved in the pathogenesis of hepatocellular steatosis.
Asunto(s)
Hígado Graso/genética , Hepatocitos/metabolismo , Obesidad/genética , PPAR gamma/genética , Fosfotransferasas (Aceptor de Grupo Alcohol)/deficiencia , Animales , Dieta Alta en Grasa/efectos adversos , Grasas de la Dieta/efectos adversos , Hígado Graso/metabolismo , Hígado Graso/patología , Regulación de la Expresión Génica , Hepatocitos/efectos de los fármacos , Hepatocitos/patología , Lisofosfolípidos/farmacología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Obesos , Obesidad/etiología , Obesidad/metabolismo , Obesidad/patología , PPAR gamma/antagonistas & inhibidores , PPAR gamma/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Receptores de Lisoesfingolípidos/genética , Receptores de Lisoesfingolípidos/metabolismo , Transducción de Señal , Esfingosina/análogos & derivados , Esfingosina/farmacología , Receptores de Esfingosina-1-Fosfato , Serina-Treonina Quinasas TOR/genética , Serina-Treonina Quinasas TOR/metabolismo , Transcripción GenéticaRESUMEN
CD8 T-cell responses to liver-expressed antigens range from deletional tolerance to full effector differentiation resulting in overt hepatotoxicity. The reasons for these heterogeneous outcomes are not well understood. To identify factors that govern the fate of CD8 T cells activated by hepatocyte-expressed antigen, we exploited recombinant adenoassociated viral vectors that enabled us to vary potential parameters determining these outcomes in vivo. Our findings reveal a threshold of antigen expression within the liver as the dominant factor determining T-cell fate, irrespective of T-cell receptor affinity or antigen cross-presentation. Thus, when a low percentage of hepatocytes expressed cognate antigen, high-affinity T cells developed and maintained effector function, whereas, at a high percentage, they became functionally exhausted and silenced. Exhaustion was not irreversibly determined by initial activation, but was maintained by high intrahepatic antigen load during the early phase of the response; cytolytic function was restored when T cells primed under high antigen load conditions were transferred into an environment of low-level antigen expression. Our study reveals a hierarchy of factors dictating the fate of CD8 T cells during hepatic immune responses, and provides an explanation for the different immune outcomes observed in a variety of immune-mediated liver pathologic conditions.
Asunto(s)
Antígenos/inmunología , Linfocitos T CD8-positivos/inmunología , Regulación de la Expresión Génica/inmunología , Hepatocitos/inmunología , Hígado/inmunología , Animales , Antígenos/genética , Linfocitos T CD8-positivos/citología , Regulación de la Expresión Génica/genética , Hepatocitos/citología , Hígado/citología , Ratones , Ratones NoqueadosRESUMEN
BACKGROUND: The rates of oropharyngeal cancers such as tonsil cancers are increasing. The tumour suppressor protein Programmed Cell Death Protein 4 (PDCD4) has been implicated in the development of various human cancers and small RNAs such as microRNAs (miRNAs) can regulate its expression. However the exact regulation of PDCD4 by multiple miRNAs in oropharyngeal squamous cell carcinoma (SCC) is not well understood. RESULTS: Using two independent oropharyngeal SCC cohorts with a focus on the tonsillar region, we identified a miRNA profile differentiating SCC tissue from normal. Both miR-21 and miR-499 were highly expressed in tonsil SCC tissues displaying a loss of PDCD4. Interestingly, expression of the miRNA machinery, Dicer1, Drosha, DDX5 (Dead Box Helicase 5) and DGCR8 (DiGeorge Syndrome Critical Region Gene 8) were all elevated by greater than 2 fold in the tonsil SCC tissue. The 3'UTR of PDCD4 contains three binding-sites for miR-499 and one for miR-21. Using a wild-type and truncated 3'UTR of PDCD4, we demonstrated that the initial suppression of PDCD4 was mediated by miR-21 whilst sustained suppression was mediated by miR-499. Moreover the single miR-21 site was able to elicit the same magnitude of suppression as the three miR-499 sites. CONCLUSION: This study describes the regulation of PDCD4 specifically in tonsil SCC by miR-499 and miR-21 and has documented the loss of PDCD4 in tonsil SCCs. These findings highlight the complex interplay between miRNAs and tumour suppressor gene regulation and suggest that PDCD4 loss may be an important step in tonsillar carcinogenesis.
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Proteínas Reguladoras de la Apoptosis/biosíntesis , Carcinoma de Células Escamosas/genética , MicroARNs/genética , Neoplasias Orofaríngeas/genética , Proteínas de Unión al ARN/biosíntesis , Proteínas Reguladoras de la Apoptosis/genética , Carcinogénesis/genética , Carcinoma de Células Escamosas/patología , Línea Celular Tumoral , Proliferación Celular/genética , ARN Helicasas DEAD-box/biosíntesis , Regulación Neoplásica de la Expresión Génica , Humanos , MicroARNs/biosíntesis , Neoplasias Orofaríngeas/patología , Proteínas de Unión al ARN/genética , Ribonucleasa III/biosíntesisRESUMEN
Naive T cell activation is normally restricted to the lymphoid organs, in part because of their limited ability to migrate into the parenchyma of peripheral tissues. The liver vasculature is unique, however, and circulating leukocytes within the hepatic sinusoids have direct access to liver-resident cells, which include an abundant population of Kupffer cells. It is well accepted that recognition of cognate Ag within the liver leads to naive CD8(+) T cell activation in situ, but it is unclear whether the liver also supports naive CD4(+) T cell activation. In this study, we show that naive CD4(+) T cells can be activated to proliferate in the liver when cognate Ag expression is induced in hepatocytes by recombinant adeno-associated viral vectors. Ag-specific retention and activation of naive CD4(+) T cells within the liver are independent of lymphoid tissues but dependent on a clodronate liposome-sensitive population of liver-resident phagocytic cells. To our knowledge, this study provides the first unequivocal evidence that naive CD4(+) T cells can be activated in a nonlymphoid organ. It also gives critical insight into how CD4(+) T cells specific for Ag expressed in the liver are recruited to participate in protective or pathological responses during hepatotropic infections and autoimmune liver disease.
Asunto(s)
Enfermedades Autoinmunes/inmunología , Linfocitos T CD4-Positivos/inmunología , Macrófagos del Hígado/inmunología , Hepatopatías/inmunología , Hígado/inmunología , Animales , Enfermedades Autoinmunes/genética , Enfermedades Autoinmunes/patología , Conservadores de la Densidad Ósea/farmacología , Linfocitos T CD4-Positivos/patología , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/patología , Ácido Clodrónico/farmacología , Macrófagos del Hígado/patología , Liposomas , Hígado/patología , Hepatopatías/genética , Hepatopatías/patología , Activación de Linfocitos , Ratones , Ratones TransgénicosRESUMEN
Cellular junctions are essential to the normal functioning of the endothelium and control angiogenesis, tissue leak, and inflammation. From a screen of micro RNAs (miRNAs) altered in in vitro angiogenesis, we selected a subset predicted to target junctional molecules. MiR-27a was rapidly downregulated upon stimulation of in vitro angiogenesis, and its level of expression is reduced in neovessels in vivo. The downregulation of miR-27a was essential for angiogenesis because ectopic expression of miR-27a blocked capillary tube formation and angiogenesis. MiR-27a targets the junctional, endothelial-specific cadherin, VE-cadherin. Consistent with this, vascular permeability to vascular endothelial growth factor in mice is reduced by administration of a general miR-27 inhibitor. To determine that VE-cadherin was the dominant target of miR-27a function, we used a novel technology with "Blockmirs," inhibitors that bind to the miR-27 binding site in VE-cadherin. The Blockmir CD5-2 demonstrated specificity for VE-cadherin and inhibited vascular leak in vitro and in vivo. Furthermore, CD5-2 reduced edema, increased capillary density, and potently enhanced recovery from ischemic limb injury in mice. The Blockmir technology offers a refinement in the use of miRNAs, especially for therapy. Further, targeting of endothelial junctional molecules by miRNAs has clinical potential, especially in diseases associated with vascular leak.
Asunto(s)
Antígenos CD/metabolismo , Cadherinas/metabolismo , Regulación de la Expresión Génica , MicroARNs/metabolismo , Animales , Sitios de Unión , Permeabilidad Capilar , Edema/patología , Células HEK293 , Células Endoteliales de la Vena Umbilical Humana , Humanos , Isquemia/patología , Cirrosis Hepática/patología , Ratones , Ratones Endogámicos C57BL , MicroARNs/antagonistas & inhibidores , Neovascularización PatológicaRESUMEN
BACKGROUND & AIMS: Epithelial-mesenchymal transition (EMT) has been implicated in the processes of embryogenesis, tissue fibrosis and carcinogenesis. Transforming growth factor-ß (TGF-ß) has been identified as a key driver of EMT and plays a key role in the pathogenesis of cirrhosis and hepatocellular carcinoma (HCC). The aim was to identify microRNA (miR) expression in TGF-ß-induced hepatocyte EMT. METHODS: We treated a human hepatocyte cell line PH5CH8 with TGF-ß to induce an EMT-like change in phenotype and then identified dysregulated miRs using TaqMan Low Density Arrays. MiR expression was altered using miR-181a mimic and inhibitor in the same system and gene changes were identified using TaqMan gene arrays. MiR-181a gene expression was measured in human and mouse cirrhotic or HCC liver tissue samples. Gene changes were identified in rAAV-miR-181a-expressing mouse livers using TaqMan gene arrays. RESULTS: We identified miR-181a as a miR that was significantly up-regulated in response to TGF-ß treatment. Over-expression of a miR-181a mimic induced an in vitro EMT-like change with a phenotype similar to that seen with TGF-ß treatment alone and was reversed using a miR-181a inhibitor. MiR-181a was shown to be up-regulated in experimental and human cirrhotic and HCC tissue. Mouse livers expressing rAAV-miR-181a showed genetic changes associated with TGF-ß signalling and EMT. CONCLUSIONS: MiR-181a had a direct effect in inducing hepatocyte EMT and was able to replace TGF-ß-induced effects in vitro. MiR-181a was over-expressed in cirrhosis and HCC and is likely to play a role in disease pathogenesis.
Asunto(s)
Carcinoma Hepatocelular/metabolismo , Transición Epitelial-Mesenquimal/fisiología , Regulación de la Expresión Génica/efectos de los fármacos , Hepatocitos/fisiología , Cirrosis Hepática/metabolismo , Neoplasias Hepáticas/metabolismo , MicroARNs/metabolismo , Animales , Línea Celular , Hepatocitos/citología , Humanos , Técnicas In Vitro , Ratones , MicroARNs/genética , Factor de Crecimiento Transformador beta/metabolismo , Factor de Crecimiento Transformador beta/farmacologíaRESUMEN
Hepatocellular carcinoma (HCC) is the fourth-leading cause of cancer-related death worldwide. Due to the high mortality rate in HCC patients, discovering and developing novel systemic treatment options for HCC is a vital unmet medical need. Among the numerous molecular alterations in HCCs, microRNAs (miRNAs) have been increasingly recognised to play critical roles in hepatocarcinogenesis. We and others have recently revealed that members of the microRNA-181 (miR-181) family were up-regulated in some, though not all, human cirrhotic and HCC tissues-this up-regulation induced epithelial-mesenchymal transition (EMT) in hepatocytes and tumour cells, promoting HCC progression. MiR-181s play crucial roles in governing the fate and function of various cells, such as endothelial cells, immune cells, and tumour cells. Previous reviews have extensively covered these aspects in detail. This review aims to give some insights into miR-181s, their targets and roles in modulating signal transduction pathways, factors regulating miR-181 expression and function, and their roles in HCC.
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Carcinoma Hepatocelular , Neoplasias Hepáticas , MicroARNs , Humanos , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patología , Carcinoma Hepatocelular/metabolismo , MicroARNs/genética , MicroARNs/metabolismo , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patología , Neoplasias Hepáticas/metabolismo , Regulación Neoplásica de la Expresión Génica , Transición Epitelial-Mesenquimal/genética , Transducción de Señal , AnimalesRESUMEN
BACKGROUND: The learning active subnetworks problem involves finding subnetworks of a bio-molecular network that are active in a particular condition. Many approaches integrate observation data (e.g., gene expression) with the network topology to find candidate subnetworks. Increasingly, pathway databases contain additional annotation information that can be mined to improve prediction accuracy, e.g., interaction mechanism (e.g., transcription, microRNA, cleavage) annotations. We introduce a mechanism-based approach to active subnetwork recovery which exploits such annotations. We suggest that neighboring interactions in a network tend to be co-activated in a way that depends on the "correlation" of their mechanism annotations. e.g., neighboring phosphorylation and de-phosphorylation interactions may be more likely to be co-activated than neighboring phosphorylation and covalent bonding interactions. RESULTS: Our method iteratively learns the mechanism correlations and finds the most likely active subnetwork. We use a probabilistic graphical model with a Markov Random Field component which creates dependencies between the states (active or non-active) of neighboring interactions, that incorporates a mechanism-based component to the function. We apply a heuristic-based EM-based algorithm suitable for the problem. We validated our method's performance using simulated data in networks downloaded from GeneGO against the same approach without the mechanism-based component, and two other existing methods. We validated our methods performance in correctly recovering (1) the true interaction states, and (2) global network properties of the original network against these other methods. We applied our method to networks generated from time-course gene expression studies in angiogenesis and lung organogenesis and validated the findings from a biological perspective against current literature. CONCLUSIONS: The advantage of our mechanism-based approach is best seen in networks composed of connected regions with a large number of interactions annotated with a subset of mechanisms, e.g., a regulatory region of transcription interactions, or a cleavage cascade region. When applied to real datasets, our method recovered novel and biologically meaningful putative interactions, e.g., interactions from an integrin signaling pathway using the angiogenesis dataset, and a group of regulatory microRNA interactions in an organogenesis network.
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Redes Reguladoras de Genes , Neovascularización Fisiológica/genética , Organogénesis/genética , Algoritmos , Animales , Ratones , Modelos Estadísticos , Mapeo de Interacción de Proteínas , Transducción de SeñalRESUMEN
Vessels that encapsulate tumour clusters (VETC) is a distinct histologic vascular pattern associated with a novel mechanism of metastasis. First described in human cancers in 2004, its prevalence and prognostic significance in hepatocellular carcinoma (HCC) has only been appreciated in the past decade with a rapidly increasing body of literature. A robust biomarker of aggressive disease, the VETC pattern is easy to recognise but relies on histologic examination of tumour tissue for its diagnosis. Radiological recognition of the VETC pattern is an area of active research and is becoming increasingly accurate. As a prognostic marker, VETC has consistently proven to be an independent predictor of disease recurrence and overall survival in patients with HCC undergoing resection and liver transplantation. It can also guide treatment by predicting response to other therapies such as transarterial chemoembolisation and sorafenib. Without prospective randomised-controlled trials or routine evaluation of VETC in clinical practice, there are currently no firm treatment recommendations for VETC-positive tumours, although some perspectives are provided in this review based on the latest knowledge of their pathogenesis - a complex interplay between tumour angiogenesis and the immune microenvironment. Nevertheless, VETC has great potential as a future biomarker that could take us one step closer to precision medicine for HCC.
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Introduction: Liver cancers exhibit abnormal (leaky) vasculature, hypoxia and an immunosuppressive microenvironment. Normalization of tumor vasculature is an emerging approach to treat many cancers. Blockmir CD5-2 is a novel oligonucleotide-based inhibitor of the miR-27a interaction with VE-Cadherin, the endothelial-specific cadherin. The combination of a vasoactive medication with inhibition of immune checkpoints such as programmed cell death protein 1 (PD1) has been shown to be effective in treating liver cancer in humans. We aimed to study the effect of CD5-2 combined with checkpoint inhibition (using an antibody against PD1) on liver tumor growth, vasculature and immune infiltrate in the diethylnitrosamine (DEN)-induced liver tumor mouse model. Methods: We first analyzed human miR-27a and VE-Cadherin expression data from The Cancer Genome Atlas for hepatocellular carcinoma. CD5-2 and/or anti-PD1 antibody were given to the DEN-treated mice from age 7-months until harvest at age 9-months. Tumor and non-tumor liver tissues were analyzed using histology, immunohistochemistry, immunofluorescence and scanning electron microscopy. Results: Human data showed high miR-27a and low VE-Cadherin were both significantly associated with poorer prognosis. Mice treated with CD5-2 plus anti-PD1 antibody had significantly smaller liver tumors (50% reduction) compared to mice treated with either agent alone, controls, or untreated mice. There was no difference in tumor number. Histologically, tumors in CD5-2-treated mice had less leaky vessels with higher VE-Cadherin expression and less tumor hypoxia compared to non-CD5-2-treated mice. Only tumors in the combination CD5-2 plus anti-PD1 antibody group exhibited a more favorable immune infiltrate (significantly higher CD3+ and CD8+ T cells and lower Ly6G+ neutrophils) compared to tumors from other groups. Discussion: CD5-2 normalized tumor vasculature and reduced hypoxia in DEN-induced liver tumors. CD5-2 plus anti-PD1 antibody reduced liver tumor size possibly by altering the immune infiltrate to a more immunosupportive one.
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Neoplasias Hepáticas , MicroARNs , Humanos , Ratones , Animales , Lactante , Dietilnitrosamina/toxicidad , Neoplasias Hepáticas/inducido químicamente , Neoplasias Hepáticas/tratamiento farmacológico , Neoplasias Hepáticas/genética , MicroARNs/genética , MicroARNs/metabolismo , Hipoxia , Microambiente TumoralRESUMEN
Alzheimer's disease (AD) is an age-related disease, with loss of integrity of the blood-brain barrier (BBB) being an early feature. Cellular senescence is one of the reported nine hallmarks of aging. Here, we show for the first time the presence of senescent cells in the vasculature in AD patients and mouse models of AD. Senescent endothelial cells and pericytes are present in APP/PS1 transgenic mice but not in wild-type littermates at the time of amyloid deposition. In vitro, senescent endothelial cells display altered VE-cadherin expression and loss of cell junction formation and increased permeability. Consistent with this, senescent endothelial cells in APP/PS1 mice are present at areas of vascular leak that have decreased claudin-5 and VE-cadherin expression confirming BBB breakdown. Furthermore, single cell sequencing of endothelial cells from APP/PS1 transgenic mice confirms that adhesion molecule pathways are among the most highly altered pathways in these cells. At the pre-plaque stage, the vasculature shows significant signs of breakdown, with a general loss of VE-cadherin, leakage within the microcirculation, and obvious pericyte perturbation. Although senescent vascular cells were not directly observed at sites of vascular leak, senescent cells were close to the leak area. Thus, we would suggest in AD that there is a progressive induction of senescence in constituents of the neurovascular unit contributing to an increasing loss of vascular integrity. Targeting the vasculature early in AD, either with senolytics or with drugs that improve the integrity of the BBB may be valid therapeutic strategies.
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Enfermedad de Alzheimer , Barrera Hematoencefálica , Humanos , Ratones , Animales , Barrera Hematoencefálica/metabolismo , Enfermedad de Alzheimer/metabolismo , Células Endoteliales , Ratones Transgénicos , EnvejecimientoRESUMEN
Cellular senescence is a mechanism to inhibit the growth of mammalian cells after oncogenic activation, or in response to damage or stress. We describe here the identification of a novel gene, SENEX, that regulates stress induced premature senescence pathways in endothelial cells (ECs) involving p16(INK4a) and retinoblastoma protein activation. Endogenous levels of SENEX remain unchanged during replicative senescence but are regulated by H(2)O(2)-mediated stress. In contrast to that previously described for senescence in other cell types, the SENEX induced senescent ECs are profoundly anti-inflammatory. The cells are resistant to tumor necrosis factor (TNF)α-induced apoptosis, adhesion of neutrophils and mononuclear cells, and the surface (but not cytoplasmic) expression of endothelial leukocyte adhesion molecule 1 and vascular cell adhesion molecule 1. Furthermore they are resistant to thrombin induced vascular leak. Senescent ECs such as those lining atherosclerotic lesions may therefore function to limit the inflammatory response. SENEX is also essential for EC survival since depletion either ectopically by siRNA or by high- dose H(2)O(2) treatment causes apoptosis. Together, these findings expand our understanding of the role of senescence in the vasculature and identify SENEX as a fulcrum for driving the resultant phenotype of the endothelium after activation.
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Senescencia Celular/genética , Células Endoteliales/citología , Estrés Oxidativo/genética , Apoptosis/efectos de los fármacos , Apoptosis/genética , Adhesión Celular , Supervivencia Celular/genética , Células Cultivadas , Inhibidor p16 de la Quinasa Dependiente de Ciclina/metabolismo , Células Endoteliales/metabolismo , Expresión Génica/efectos de los fármacos , Humanos , Peróxido de Hidrógeno/toxicidad , Inflamación/genética , Inflamación/metabolismo , Inflamación/patología , Inflamación/prevención & control , Neovascularización Fisiológica/genética , Fenotipo , ARN Interferente Pequeño/genética , Transducción de Señal , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
Neutrophil extravasation, a critical component of innate immunity must be tightly regulated to prevent inadvertent or prolonged inflammation and subsequent tissue damage. We have shown previously that endothelial ERK1/2 signaling essential for neutrophil transendothelial migration is induced by a soluble factor produced by activated neutrophils. In this study, we demonstrate that the soluble neutrophil factor is a truncated form of annexin A1 (AnxA1) that can be generated by calpain 1 cleavage of the N terminus, thus identifying a novel proinflammatory function to AnxA1. In contrast, neither the full-length protein nor the N-terminal 26 aa peptide, previously shown to be antiinflammatory, were able to activate Erk. Our data suggest that two different fragments of AnxA1 have opposing functions in inflammation. We also provide evidence that C-terminal AnxA1 functions by increasing ICAM1 clustering around adherent neutrophils to anchor them to the endothelium and promote transmigration through the transcellular route.
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Anexina A1/fisiología , Movimiento Celular/inmunología , Endotelio Vascular/metabolismo , Mediadores de Inflamación/fisiología , Activación Neutrófila/inmunología , Neutrófilos/metabolismo , Fragmentos de Péptidos/fisiología , Secuencia de Aminoácidos , Anexina A1/metabolismo , Calpaína/metabolismo , Línea Celular , Endotelio Vascular/enzimología , Endotelio Vascular/patología , Humanos , Mediadores de Inflamación/metabolismo , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Datos de Secuencia Molecular , Neutrófilos/patología , Fragmentos de Péptidos/metabolismoRESUMEN
Circulating endothelial progenitor cells (EPCs) are incorporated into foci of neovascularization where they undergo differentiation to mature endothelial cells (ECs). We show here that the enzyme sphingosine kinase-1 (SK-1) regulates the rate and direction of EPC differentiation without effect on the hematopoietic compartment. EPCs have high levels of SK-1 activity, which diminishes with differentiation and is, at least partially, responsible for maintaining their EPC phenotype. EPCs from SK-1 knockout mice form more adherent EC units and acquire a mature EC phenotype more rapidly. Conversely, EPCs from mice overexpressing SK-1 in the EC compartment are retarded in their differentiation. Exogenous regulation of SK-1 levels in normal EPCs, by genetic and pharmacologic means, including the immunomodulating drug FTY720, recapitulates these effects on EC differentiation. SK-1 knockout mice have higher levels of circulating EPCs, an exaggerated response to erythropoietin-induced EPC mobilization, and, in a mouse model of kidney ischemia reperfusion injury, exhibit a recovery similar to that of ischemic mice administered exogenous EPCs. Thus, SK-1 is a critical player in EPC differentiation into EC pointing to the potential utility of SK-1 modifying agents in the specific manipulation of endothelial development and repair.