RESUMO
BACKGROUND AND AIMS: In the treatment of chronic hepatitis B (CHB) infection, stimulation of innate immunity may lead to hepatitis B virus (HBV) cure. Alpha-kinase 1 (ALPK1) is a pattern recognition receptor (PRR) that activates the NF-κB pathway and stimulates innate immunity. Here we characterized the preclinical anti-HBV efficacy of DF-006, an orally active agonist of ALPK1 currently in clinical development for CHB. APPROACH AND RESULTS: In adeno-associated virus (AAV)-HBV mouse models and primary human hepatocytes (PHHs) infected with HBV, we evaluated the antiviral efficacy of DF-006. In the mouse models, DF-006 rapidly reduced serum HBV DNA, hepatitis B surface antigen, and hepatitis B e antigen levels using doses as low as 0.08 µg/kg, 1 µg/kg, and 5 µg/kg, respectively. DF-006 in combination with the HBV nucleoside reverse transcriptase inhibitor, entecavir, further reduced HBV DNA. Antiviral efficacy in mice was associated with an increase in immune cell infiltration and decrease of hepatitis B core antigen, encapsidated pregenomic RNA, and covalently closed circular DNA in liver. At subnanomolar concentrations, DF-006 also showed anti-HBV efficacy in PHH with significant reductions of HBV DNA. Following dosing with DF-006, there was upregulation of NF-κB-targeted genes that are involved in innate immunity. CONCLUSION: DF-006 was efficacious in mouse and PHH models of HBV without any indications of overt toxicity. In mice, DF-006 localized primarily to the liver where it potently activated innate immunity. The transcriptional response in mouse liver provides insights into mechanisms that mediate anti-HBV efficacy by DF-006.
Assuntos
Hepatite B Crônica , Hepatite B , Humanos , Camundongos , Animais , DNA Viral , NF-kappa B/metabolismo , Hepatócitos/metabolismo , Vírus da Hepatite B/genética , Antivirais/farmacologia , Antivirais/uso terapêuticoRESUMO
Angiogenesis and vascular remodeling are essential for the establishment of vascular networks during organogenesis. Here we show that the Hippo signaling pathway effectors YAP and TAZ are required, in a gene dosage-dependent manner, for the proliferation and migration of vascular endothelial cells (ECs) during retinal angiogenesis. Intriguingly, nuclear translocation of YAP and TAZ induced by Lats1/2-deletion blocked endothelial migration and phenocopied Yap/Taz-deficient mutants. Furthermore, overexpression of a cytoplasmic form of YAP (YAPS127D) partially rescued the migration defects caused by loss of YAP and TAZ function. Finally, we found that cytoplasmic YAP positively regulated the activity of the small GTPase CDC42, deletion of which caused severe defects in endothelial migration. These findings uncover a previously unrecognized role of cytoplasmic YAP/TAZ in promoting cell migration by activating CDC42 and provide insight into how Hippo signaling in ECs regulates angiogenesis.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Movimento Celular , Endotélio Vascular/citologia , Neovascularização Fisiológica , Fosfoproteínas/fisiologia , Fatores de Transcrição/fisiologia , Proteína cdc42 de Ligação ao GTP/fisiologia , Aciltransferases , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Proteínas de Ciclo Celular , Proliferação de Células , Endotélio Vascular/fisiologia , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Camundongos , Camundongos Knockout , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Transdução de Sinais , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteínas de Sinalização YAPRESUMO
Endothelial cells respond to molecular and physical forces in development and vascular homeostasis. Deregulation of endothelial responses to flow-induced shear is believed to contribute to many aspects of cardiovascular diseases including atherosclerosis. However, how molecular signals and shear-mediated physical forces integrate to regulate vascular patterning is poorly understood. Here we show that endothelial non-canonical Wnt signalling regulates endothelial sensitivity to shear forces. Loss of Wnt5a/Wnt11 renders endothelial cells more sensitive to shear, resulting in axial polarization and migration against flow at lower shear levels. Integration of flow modelling and polarity analysis in entire vascular networks demonstrates that polarization against flow is achieved differentially in artery, vein, capillaries and the primitive sprouting front. Collectively our data suggest that non-canonical Wnt signalling stabilizes forming vascular networks by reducing endothelial shear sensitivity, thus keeping vessels open under low flow conditions that prevail in the primitive plexus.
Assuntos
Células Endoteliais/fisiologia , Estresse Mecânico , Remodelação Vascular , Via de Sinalização Wnt , Animais , Linhagem Celular , Movimento Celular , Polaridade Celular , Regulação da Expressão Gênica , CamundongosRESUMO
The developing lens is a powerful system for investigating the molecular basis of inductive tissue interactions and for studying cataract, the leading cause of blindness. The formation of tightly controlled cell-cell adhesions and cell-matrix junctions between lens epithelial (LE) cells, between lens fiber (LF) cells, and between these two cell populations enables the vertebrate lens to adopt a highly ordered structure and acquire optical transparency. Adhesion molecules are thought to maintain this ordered structure, but little is known about their identity or interactions. Cysteine-rich motor neuron 1 (Crim1), a type I transmembrane protein, is strongly expressed in the developing lens and its mutation causes ocular disease in both mice and humans. How Crim1 regulates lens morphogenesis is not understood. We identified a novel ENU-induced hypomorphic allele of Crim1, Crim1(glcr11), which in the homozygous state causes cataract and microphthalmia. Using this and two other mutant alleles, Crim1(null) and Crim1(cko), we show that the lens defects in Crim1 mouse mutants originate from defective LE cell polarity, proliferation and cell adhesion. Crim1 adhesive function is likely to be required for interactions both between LE cells and between LE and LF cells. We show that Crim1 acts in LE cells, where it colocalizes with and regulates the levels of active ß1 integrin and of phosphorylated FAK and ERK. The RGD and transmembrane motifs of Crim1 are required for regulating FAK phosphorylation. These results identify an important function for Crim1 in the regulation of integrin- and FAK-mediated LE cell adhesion during lens development.
Assuntos
Receptores de Proteínas Morfogenéticas Ósseas/metabolismo , Cristalino/citologia , Animais , Receptores de Proteínas Morfogenéticas Ósseas/genética , Linhagem Celular , Regulação da Expressão Gênica no Desenvolvimento , Imuno-Histoquímica , Marcação In Situ das Extremidades Cortadas , Cristalino/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Organogênese/genética , Organogênese/fisiologia , Fosforilação , Transdução de Sinais/fisiologiaRESUMO
Colobomatous macrophthalmia with microcornea syndrome (MACOM, Online Mendelian Inheritance in Man (OMIM) 602499) is an autosomal dominantly inherited malformation of the eye, which is characterized by microcornea with increased axial length, coloboma of the iris and of the optic disc, and severe myopia. We performed whole-exome sequencing (WES) in two affected individuals from the 2p23-p16-linked MACOM family, which includes 13 affected individuals in 3 generations. As no shared novel variation was found on the linked haplotype, we performed copy number variation (CNV) analysis by comparing the coverage of all exons in the WES data sets of the 2 patients with the coverage of 26 control exomes. We identified a heterozygous deletion predicted to span 22 kb including exons 14-17 of CRIM1 (cysteine-rich transmembrane bone morphogenetic protein (BMP) regulator 1). Quantitative PCR (qPCR) analysis confirmed the deletion, which was present in 11 affected individuals. Split-read analysis of WES data followed by breakpoint PCR and Sanger sequencing determined both breakpoints flanked by a 4-bp microhomology (CTTG). In the mouse, Crim1 is a growth-factor-binding protein with pleiotropic roles in the development of multiple organs, including the eye. To investigate the role of Crim1 during eye development in mice, we crossed a Crim1(flox) mouse line with the Ap2α-cre mouse line, which expresses Cre in the head surface ectoderm. Strikingly, we observed alterations of eye development in homozygous mice leading to severe anatomical and morphological changes overlapping with the anomalies observed in MACOM patients. Taken together, these findings identify CRIM1 as the causative gene for MACOM syndrome and emphasize the importance of CRIM1 in eye development.
Assuntos
Receptores de Proteínas Morfogenéticas Ósseas/metabolismo , Doenças da Córnea/genética , Anormalidades do Olho/genética , Olho/crescimento & desenvolvimento , Haploinsuficiência , Proteínas de Membrana/metabolismo , Adulto , Animais , Sequência de Bases , Receptores de Proteínas Morfogenéticas Ósseas/genética , Doenças da Córnea/metabolismo , Doenças da Córnea/fisiopatologia , Variações do Número de Cópias de DNA , Éxons , Olho/anatomia & histologia , Olho/metabolismo , Anormalidades do Olho/metabolismo , Anormalidades do Olho/fisiopatologia , Feminino , Homozigoto , Humanos , Masculino , Proteínas de Membrana/genética , Camundongos , Dados de Sequência Molecular , Linhagem , Adulto JovemRESUMO
Angiogenesis defines the process in which new vessels grow from existing vessels. Using the mouse retina as a model system, we show that cysteine-rich motor neuron 1 (Crim1), a type I transmembrane protein, is highly expressed in angiogenic endothelial cells. Conditional deletion of the Crim1 gene in vascular endothelial cells (VECs) causes delayed vessel expansion and reduced vessel density. Based on known Vegfa binding by Crim1 and Crim1 expression in retinal vasculature, where angiogenesis is known to be Vegfa dependent, we tested the hypothesis that Crim1 is involved in the regulation of Vegfa signaling. Consistent with this hypothesis, we showed that VEC-specific conditional compound heterozygotes for Crim1 and Vegfa exhibit a phenotype that is more severe than each single heterozygote and indistinguishable from that of the conditional homozygotes. We further showed that human CRIM1 knockdown in cultured VECs results in diminished phosphorylation of VEGFR2, but only when VECs are required to rely on an autocrine source of VEGFA. The effect of CRIM1 knockdown on reducing VEGFR2 phosphorylation was enhanced when VEGFA was also knocked down. Finally, an anti-VEGFA antibody did not enhance the effect of CRIM1 knockdown in reducing VEGFR2 phosphorylation caused by autocrine signaling, but VEGFR2 phosphorylation was completely suppressed by SU5416, a small-molecule VEGFR2 kinase inhibitor. These data are consistent with a model in which Crim1 enhances the autocrine signaling activity of Vegfa in VECs at least in part via Vegfr2.
Assuntos
Receptores de Proteínas Morfogenéticas Ósseas/metabolismo , Vasos Retinianos/crescimento & desenvolvimento , Vasos Retinianos/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Alelos , Animais , Comunicação Autócrina , Receptores de Proteínas Morfogenéticas Ósseas/antagonistas & inibidores , Receptores de Proteínas Morfogenéticas Ósseas/genética , Proliferação de Células , Células Endoteliais/metabolismo , Técnicas de Silenciamento de Genes , Heterozigoto , Homozigoto , Células Endoteliais da Veia Umbilical Humana , Humanos , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Mutantes , Camundongos Transgênicos , Neovascularização Fisiológica , Pericitos/metabolismo , Fenótipo , Fosforilação , RNA Interferente Pequeno/genética , Vasos Retinianos/embriologia , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Fator A de Crescimento do Endotélio Vascular/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
Vascular patterning is critical for organ function. In the eye, there is simultaneous regression of embryonic hyaloid vasculature (important to clear the optical path) and formation of the retinal vasculature (important for the high metabolic demands of retinal neurons). These events occur postnatally in the mouse. Here we have identified a light-response pathway that regulates both processes. We show that when mice are mutated in the gene (Opn4) for the atypical opsin melanopsin, or are dark-reared from late gestation, the hyaloid vessels are persistent at 8 days post-partum and the retinal vasculature overgrows. We provide evidence that these vascular anomalies are explained by a light-response pathway that suppresses retinal neuron number, limits hypoxia and, as a consequence, holds local expression of vascular endothelial growth factor (VEGFA) in check. We also show that the light response for this pathway occurs in late gestation at about embryonic day 16 and requires the photopigment in the fetus and not the mother. Measurements show that visceral cavity photon flux is probably sufficient to activate melanopsin-expressing retinal ganglion cells in the mouse fetus. These data thus show that light--the stimulus for function of the mature eye--is also critical in preparing the eye for vision by regulating retinal neuron number and initiating a series of events that ultimately pattern the ocular blood vessels.
Assuntos
Olho/irrigação sanguínea , Olho/crescimento & desenvolvimento , Feto/efeitos da radiação , Transdução de Sinal Luminoso/efeitos da radiação , Luz , Neurônios Retinianos/efeitos da radiação , Opsinas de Bastonetes/metabolismo , Animais , Contagem de Células , Hipóxia Celular/efeitos da radiação , Olho/metabolismo , Olho/efeitos da radiação , Feminino , Feto/citologia , Feto/embriologia , Feto/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Neovascularização Patológica , Neovascularização Fisiológica/efeitos da radiação , Fótons , Células Ganglionares da Retina/citologia , Células Ganglionares da Retina/metabolismo , Células Ganglionares da Retina/efeitos da radiação , Neurônios Retinianos/citologia , Neurônios Retinianos/metabolismo , Opsinas de Bastonetes/deficiência , Opsinas de Bastonetes/genética , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
In multicellular organisms, morphogenesis is a highly coordinated process that requires dynamically regulated adhesion between cells. An excellent example of cellular morphogenesis is the formation of the neural tube from the flattened epithelium of the neural plate. Cysteine-rich motor neuron protein 1 (CRIM1) is a single-pass (type 1) transmembrane protein that is expressed in neural structures beginning at the neural plate stage. In the frog Xenopus laevis, loss of function studies using CRIM1 antisense morpholino oligonucleotides resulted in a failure of neural development. The CRIM1 knockdown phenotype was, in some cases, mild and resulted in perturbed neural fold morphogenesis. In severely affected embryos there was a dramatic failure of cell adhesion in the neural plate and complete absence of neural structures subsequently. Investigation of the mechanism of CRIM1 function revealed that it can form complexes with ß-catenin and cadherins, albeit indirectly, via the cytosolic domain. Consistent with this, CRIM1 knockdown resulted in diminished levels of cadherins and ß-catenin in junctional complexes in the neural plate. We conclude that CRIM1 is critical for cell-cell adhesion during neural development because it is required for the function of cadherin-dependent junctions.
Assuntos
Junções Intercelulares/fisiologia , Proteínas de Membrana/metabolismo , Morfogênese/fisiologia , Complexos Multiproteicos/metabolismo , Tubo Neural/embriologia , Proteínas de Xenopus/metabolismo , Xenopus laevis/embriologia , Animais , Caderinas/metabolismo , Primers do DNA/genética , Imunofluorescência , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Immunoblotting , Imunoprecipitação , Hibridização In Situ , Proteínas de Membrana/fisiologia , Morfolinos , Proteínas de Xenopus/fisiologia , beta Catenina/metabolismoRESUMO
Myeloid cells are a feature of most tissues. Here we show that during development, retinal myeloid cells (RMCs) produce Wnt ligands to regulate blood vessel branching. In the mouse retina, where angiogenesis occurs postnatally, somatic deletion in RMCs of the Wnt ligand transporter Wntless results in increased angiogenesis in the deeper layers. We also show that mutation of Wnt5a and Wnt11 results in increased angiogenesis and that these ligands elicit RMC responses via a non-canonical Wnt pathway. Using cultured myeloid-like cells and RMC somatic deletion of Flt1, we show that an effector of Wnt-dependent suppression of angiogenesis by RMCs is Flt1, a naturally occurring inhibitor of vascular endothelial growth factor (VEGF). These findings indicate that resident myeloid cells can use a non-canonical, Wnt-Flt1 pathway to suppress angiogenic branching.
Assuntos
Células Mieloides/metabolismo , Neovascularização Fisiológica/fisiologia , Retina/citologia , Transdução de Sinais , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Proteínas Wnt/metabolismo , Animais , Vasos Sanguíneos/crescimento & desenvolvimento , Células Endoteliais/metabolismo , Fibroblastos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Relacionadas a Receptor de LDL/genética , Proteínas Relacionadas a Receptor de LDL/metabolismo , Ligantes , Proteína-5 Relacionada a Receptor de Lipoproteína de Baixa Densidade , Camundongos , Receptores Acoplados a Proteínas G , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/deficiência , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/genética , Proteínas Wnt/deficiência , Proteínas Wnt/genética , Proteína Wnt-5aRESUMO
The HSulf-1 gene is an important factor that modulates the sulfation status of heparan sulfate proteoglycans (HSPGs) in the extracellular matrix, resulting in disturbance of HSPG-related signal transduction pathways. Recently, HSulf-1 has been reported to be down-regulated in several human cancers. In this study, we first cloned and characterized the 5' promoter region of the HSulf-1 gene (around 400 bp) that contained high basal promoter activity. We also found that this functional promoter region was hypermethylated in a number of human cancer cell lines. Furthermore, we found that hypermethylation in this promoter region correlated with the down-regulation of the HSulf-1 expression in human breast and gastric cancer cell lines and tissue samples. These results suggest that the promoter hypermethylation may be one of the mechanisms of the HSulf-1 gene silencing in human breast and gastric cancers. Finally, we demonstrated that the HSulf-1 promoter was more frequently (p<0.05) methylated in cell-free DNA extracted from serum samples of human breast and gastric cancer patients than that of healthy people (76.2%, 55.0% and 19.0%, respectively), indicating that detection of the HSulf-1 promoter methylation in serum samples may have clinical implications in early detection and diagnosis of human breast and gastric cancers.