RESUMEN

Rationale: Patients with hepatitis B virus-related acute-on-chronic liver failure (HBV-ACLF) have a high short-term mortality rate. Semaphorin-6B (SEMA6B) plays a crucial role in the pathogenesis of HBV-ACLF, but its molecular basis remains unclear. This study aimed to elucidate the mechanisms of SEMA6B in HBV-ACLF progression. Methods: A total of 321 subjects with HBV-ACLF, liver cirrhosis (LC), chronic hepatitis B (CHB), and normal controls (NC) from a prospective multicenter cohort were studied. 84 subjects (HBV-ACLF, n = 50; LC, n = 10; CHB, n = 10; NC, n = 14) among them underwent mRNA sequencing using peripheral blood mononuclear cells (PBMCs) to clarify the mechanisms of SEMA6B in HBV-ACLF. These mechanisms were validated through in vitro studies with hepatocytes and macrophages, as well as in vivo using SEMA6B knockout mice and mice treated with synthetic SEMA6B siRNA. Results: Transcriptome analysis of PBMCs showed that SEMA6B was among the most differentially expressed genes when comparing patients with HBV-ACLF to those with LC, CHB, or NC. ROC analysis demonstrated the reliable diagnostic value of SEMA6B for HBV-ACLF in both the sequencing cohort and an external validation cohort (AUROC = 0.9788 and 0.9026, respectively). SEMA6B levels were significantly higher in the HBV-ACLF patients, especially in non-survivors, with high expression mainly observed in macrophages and hepatocytes in liver tissue. Genes significantly associated with highly expressed SEMA6B were enriched in inflammation and apoptosis pathways in HBV-ACLF non-survivors. Overexpression of SEMA6B in macrophages activated systemic inflammatory responses, while its overexpression in hepatocytes inhibited proliferation through G0/G1 cell cycle arrest and induced apoptosis. Knocking out SEMA6B rescued mice with liver failure by improving liver functions, reducing inflammatory responses, and decreasing hepatocyte apoptosis. Transcriptome analysis of liver tissue showed that SEMA6B knockout significantly ameliorated the liver failure signature, significantly downregulating inflammation-related pathways. Importantly, therapeutic delivery of synthetic SEMA6B siRNA also improved liver function, and reduced both inflammation and hepatocyte apoptosis in mice with liver failure. Conclusion: SEMA6B, a potential diagnostic biomarker for HBV-ACLF, exacerbates liver failure through macrophage-mediated systemic inflammation and hepatocyte apoptosis. These findings highlight SEMA6B as a promising early treatment target for HBV-ACLF patients.

Asunto(s)

Insuficiencia Hepática Crónica Agudizada , Apoptosis , Virus de la Hepatitis B , Hepatitis B Crónica , Hepatocitos , Macrófagos , Ratones Noqueados , Semaforinas , Semaforinas/metabolismo , Semaforinas/genética , Animales , Humanos , Insuficiencia Hepática Crónica Agudizada/virología , Insuficiencia Hepática Crónica Agudizada/metabolismo , Ratones , Masculino , Macrófagos/metabolismo , Hepatocitos/metabolismo , Hepatocitos/virología , Hepatitis B Crónica/complicaciones , Hepatitis B Crónica/patología , Persona de Mediana Edad , Femenino , Adulto , Estudios Prospectivos , Inflamación , Leucocitos Mononucleares/metabolismo , Cirrosis Hepática/virología , Cirrosis Hepática/patología , Cirrosis Hepática/metabolismo , Cirrosis Hepática/genética , Ratones Endogámicos C57BL

RESUMEN

The sympathetic nervous system controls bodily functions including vascular tone, cardiac rhythm, and the "fight-or-flight response". Sympathetic chain ganglia develop in parallel with preganglionic motor nerves extending from the neural tube, raising the question of whether axon targeting contributes to sympathetic chain formation. Using nerve-selective genetic ablations and lineage tracing in mouse, we reveal that motor nerve-associated Schwann cell precursors (SCPs) contribute sympathetic neurons and satellite glia after the initial seeding of sympathetic ganglia by neural crest. Motor nerve ablation causes mispositioning of SCP-derived sympathoblasts as well as sympathetic chain hypoplasia and fragmentation. Sympathetic neurons in motor-ablated embryos project precociously and abnormally towards dorsal root ganglia, eventually resulting in fusion of sympathetic and sensory ganglia. Cell interaction analysis identifies semaphorins as potential motor nerve-derived signaling molecules regulating sympathoblast positioning and outgrowth. Overall, central innervation functions both as infrastructure and regulatory niche to ensure the integrity of peripheral ganglia morphogenesis.

Asunto(s)

Ganglios Simpáticos , Neuronas Motoras , Cresta Neural , Células de Schwann , Sistema Nervioso Simpático , Animales , Sistema Nervioso Simpático/embriología , Ratones , Neuronas Motoras/fisiología , Células de Schwann/metabolismo , Cresta Neural/citología , Cresta Neural/metabolismo , Ganglios Simpáticos/citología , Ganglios Espinales , Semaforinas/metabolismo , Semaforinas/genética , Ratones Transgénicos , Neuroglía/metabolismo , Femenino

RESUMEN

Disruption of neocortical circuitry and architecture in humans causes numerous neurodevelopmental disorders. Neocortical cytoarchitecture is orchestrated by various transcription factors such as Satb2 that control target genes during strict time windows. In humans, mutations of SATB2 cause SATB2 Associated Syndrome (SAS), a multisymptomatic syndrome involving epilepsy, intellectual disability, speech delay, and craniofacial defects. Here we show that Satb2 controls neuronal migration and callosal axonal outgrowth during murine neocortical development by inducing the expression of the GPI-anchored protein, Semaphorin 7A (Sema7A). We find that Sema7A exerts this biological activity by heterodimerizing in cis with the transmembrane semaphorin, Sema4D. We could also observe that heterodimerization with Sema7A promotes targeting of Sema4D to the plasma membrane in vitro. Finally, we report an epilepsy-associated de novo mutation in Sema4D (Q497P) that inhibits normal glycosylation and plasma membrane localization of Sema4D-associated complexes. These results suggest that neuronal use of semaphorins during neocortical development is heteromeric, and a greater signaling complexity exists than was previously thought.

Asunto(s)

Antígenos CD , Membrana Celular , Proteínas de Unión a la Región de Fijación a la Matriz , Neocórtex , Neuronas , Multimerización de Proteína , Semaforinas , Factores de Transcripción , Semaforinas/metabolismo , Semaforinas/genética , Animales , Neocórtex/metabolismo , Membrana Celular/metabolismo , Humanos , Ratones , Proteínas de Unión a la Región de Fijación a la Matriz/metabolismo , Proteínas de Unión a la Región de Fijación a la Matriz/genética , Antígenos CD/metabolismo , Antígenos CD/genética , Factores de Transcripción/metabolismo , Factores de Transcripción/genética , Neuronas/metabolismo , Mutación , Movimiento Celular , Axones/metabolismo , Epilepsia/metabolismo , Epilepsia/genética , Cuerpo Calloso/metabolismo , Células HEK293 , Glicosilación , Masculino , Femenino , Ratones Endogámicos C57BL

RESUMEN

The microvascular system consists of two cell types: endothelial and mural (pericytes and vascular smooth muscle cells; VSMCs) cells. Communication between endothelial and mural cells plays a pivotal role in the maintenance of vascular homeostasis; however, in vivo molecular and cellular mechanisms underlying mural cell development remain unclear. In this study, we found that macrophages played a crucial role in TGFß-dependent pericyte-to-VSMC differentiation during retinal vasculature development. In mice with constitutively active Foxo1 overexpression, substantial accumulation of TGFß1-producing macrophages and pericytes around the angiogenic front region was observed. Additionally, the TGFß-SMAD pathway was activated in pericytes adjacent to macrophages, resulting in excess ectopic α-smooth muscle actin-positive VSMCs. Furthermore, we identified endothelial SEMA3C as an attractant for macrophages. In vivo neutralization of SEMA3C rescued macrophage accumulation and ectopic VSMC phenotypes in the mice, as well as drug-induced macrophage depletion. Therefore, macrophages play an important physiological role in VSMC development via the FOXO1-SEMA3C pathway.

Asunto(s)

Proteína Forkhead Box O1 , Macrófagos , Músculo Liso Vascular , Miocitos del Músculo Liso , Semaforinas , Animales , Macrófagos/metabolismo , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/citología , Ratones , Semaforinas/metabolismo , Semaforinas/genética , Proteína Forkhead Box O1/metabolismo , Proteína Forkhead Box O1/genética , Miocitos del Músculo Liso/metabolismo , Miocitos del Músculo Liso/citología , Pericitos/metabolismo , Pericitos/citología , Diferenciación Celular , Transducción de Señal , Vasos Retinianos/metabolismo , Factores de Transcripción Forkhead/metabolismo , Factores de Transcripción Forkhead/genética , Factor de Crecimiento Transformador beta1/metabolismo , Ratones Endogámicos C57BL

RESUMEN

In a developing nervous system, axonal arbors often undergo complex rearrangements before neural circuits attain their final innervation topology. In the lateral line sensory system of the zebrafish, developing sensory axons reorganize their terminal arborization patterns to establish precise neural microcircuits around the mechanosensory hair cells. However, a quantitative understanding of the changes in the sensory arbor morphology and the regulators behind the microcircuit assembly remain enigmatic. Here, we report that Semaphorin7A (Sema7A) acts as an important mediator of these processes. Utilizing a semi-automated three-dimensional neurite tracing methodology and computational techniques, we have identified and quantitatively analyzed distinct topological features that shape the network in wild-type and Sema7A loss-of-function mutants. In contrast to those of wild-type animals, the sensory axons in Sema7A mutants display aberrant arborizations with disorganized network topology and diminished contacts to hair cells. Moreover, ectopic expression of a secreted form of Sema7A by non-hair cells induces chemotropic guidance of sensory axons. Our findings propose that Sema7A likely functions both as a juxtracrine and as a secreted cue to pattern neural circuitry during sensory organ development.

Asunto(s)

Sistema de la Línea Lateral , Semaforinas , Pez Cebra , Animales , Semaforinas/metabolismo , Semaforinas/genética , Sistema de la Línea Lateral/embriología , Proteínas de Pez Cebra/metabolismo , Proteínas de Pez Cebra/genética , Axones/fisiología , Axones/metabolismo , Red Nerviosa/fisiología

RESUMEN

BACKGROUND: Metastatic prostate cancer is a leading cause of cancer-related morbidity and mortality in men, yet the underlying molecular mechanisms are poorly understood. Plexins are transmembrane receptors for semaphorins with divergent roles in many forms of cancer. We recently found that a single clinically relevant specific amino acid change (Proline1597Leucine, (P1597L)), found in metastatic deposits of prostate cancer patients, converts PlexinB1 from a metastasis suppressor to a gene that drives prostate cancer metastasis in vivo. However, the mechanism by which PlexinB1(P1597L) promotes metastasis is not known. METHODS: Pull down assays using GST-RalGDS or -GSTRaf1-RBD were used to reveal the effect of mutant or wild-type PlexinB1 expression on Rap and Ras activity respectively. Protein-protein interactions were assessed in GST pulldown assays, Akt/ERK phosphorylation by immunoblotting and protein stability by treatment with cycloheximide. Rho/ROCK activity was monitored by measuring MLC2 phosphorylation and actin stress fiber formation. PlexinB1 function was measured using cell-collapse assays. RESULTS: We show here that the single clinically relevant P1597L amino acid change converts PlexinB1 from a repressor of Ras to a Ras activator. The PlexinB1(P1597L) mutation inhibits the RapGAP activity of PlexinB1, promoting a significant increase in Ras activity. The P1597L mutation also blocks PlexinB1-mediated reduction in Rho/ROCK activity, restraining the decrease in MLC2 phosphorylation and actin stress fiber formation induced by overexpression of wild-type PlexinB1. PlexinB1(P1597L) has little effect on the interaction of PlexinB1 with small GTPases or receptor tyrosine kinases and does not inhibit PlexinB1-stimulated Akt or ERK phosphorylation. These results indicate that the mutation affects Rho signalling via the Rap/Ras pathway. The PlexinB1(P1597L) mutation inhibits morphological cell collapse induced by wild-type PlexinB1 expression, suggesting that the mutation induces a loss of an inhibitory tumour suppressor function. CONCLUSION: These results suggest that the clinically relevant P1597L mutation in PlexinB1 may transform PlexinB1 from a suppressor to a driver of metastasis in mouse models of prostate cancer by reducing the RapGAP activity of PlexinB1, leading to Ras activation. These findings highlight the PlexinB1-Rap-Ras pathway for therapeutic intervention in prostate cancer.

Asunto(s)

Proteínas del Tejido Nervioso , Neoplasias de la Próstata , Receptores de Superficie Celular , Humanos , Masculino , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismo , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/patología , Neoplasias de la Próstata/metabolismo , Línea Celular Tumoral , Mutación , Proteínas ras/genética , Proteínas ras/metabolismo , Metástasis de la Neoplasia , Animales , Fosforilación , Transducción de Señal , Ratones , Semaforinas/metabolismo , Semaforinas/genética , Proteínas de Unión al GTP rho/metabolismo , Proteínas de Unión al GTP rho/genética

RESUMEN

Semaphorin6A (Sema6A) is a repulsive guidance molecule that plays many roles in central nervous system, heart and bone development, as well as immune system responses and cell signaling in cancer. Loss of Sema6A or its receptor PlexinA2 in zebrafish leads to smaller eyes and improper retinal patterning. Here, we investigate a potential role for the Sema6A intracellular domain in zebrafish eye development and dissect which phenotypes rely on forward signaling and which rely on reverse signaling. We performed rescue experiments on zebrafish Sema6A morphants with either full-length Sema6A (Sema6A-FL) or Sema6A lacking its intracellular domain (Sema6A-ΔC). We identified that the intracellular domain is not required for eye size and retinal patterning, however it is required for retinal integrity, the number and end feet strength of Müller glia and protecting against retinal cell death. This novel function for the intracellular domain suggests a role for Sema6A reverse signaling in zebrafish eye development.

Asunto(s)

Dominios Proteicos , Retina , Semaforinas , Proteínas de Pez Cebra , Pez Cebra , Animales , Pez Cebra/metabolismo , Pez Cebra/embriología , Semaforinas/metabolismo , Semaforinas/genética , Retina/metabolismo , Proteínas de Pez Cebra/metabolismo , Proteínas de Pez Cebra/genética , Transducción de Señal , Células Ependimogliales/metabolismo , Células Ependimogliales/citología

RESUMEN

Regulated neural-metabolic-inflammatory responses are essential for maintaining physiological homeostasis. However, the molecular machinery that coordinates neural, metabolic, and inflammatory responses is largely unknown. Here, we show that semaphorin 6D (SEMA6D) coordinates anxiogenic, metabolic, and inflammatory outputs from the amygdala by maintaining synaptic homeostasis. Using genome-wide approaches, we identify SEMA6D as a pleiotropic gene for both psychiatric and metabolic traits in human. Sema6d deficiency increases anxiety in mice. When fed a high-fat diet, Sema6d-/- mice display attenuated obesity and enhanced myelopoiesis compared with control mice due to higher sympathetic activity via the ß3-adrenergic receptor. Genetic manipulation and spatial and single-nucleus transcriptomics reveal that SEMA6D in amygdalar interneurons is responsible for regulating anxiogenic and autonomic responses. Mechanistically, SEMA6D is required for synaptic maturation and γ-aminobutyric acid transmission. These results demonstrate that SEMA6D is important for the normal functioning of the neural circuits in the amygdala, coupling emotional, metabolic, and inflammatory responses.

Asunto(s)

Amígdala del Cerebelo , Semaforinas , Animales , Amígdala del Cerebelo/metabolismo , Semaforinas/metabolismo , Semaforinas/genética , Ratones , Humanos , Masculino , Ratones Noqueados , Ratones Endogámicos C57BL , Emociones/fisiología , Ansiedad/metabolismo , Inflamación/metabolismo , Dieta Alta en Grasa , Interneuronas/metabolismo , Obesidad/metabolismo

RESUMEN

More than 90% of hepatocellular carcinoma (HCC) cases develop in the presence of fibrosis or cirrhosis, making the tumor microenvironment (TME) of HCC distinctive due to the intricate interplay between cancer-associated fibroblasts (CAFs) and cancer stem cells (CSCs), which collectively regulate HCC progression. However, the mechanisms through which CSCs orchestrate the dynamics of the tumor stroma during HCC development remain elusive. Our study unveils a significant upregulation of Sema3C in fibrotic liver, HCC tissues, peripheral blood of HCC patients, as well as sorafenib-resistant tissues and cells, with its overexpression correlating with the acquisition of stemness properties in HCC. We further identify NRP1 and ITGB1 as pivotal functional receptors of Sema3C, activating downstream AKT/Gli1/c-Myc signaling pathways to bolster HCC self-renewal and tumor initiation. Additionally, HCC cells-derived Sema3C facilitated extracellular matrix (ECM) contraction and collagen deposition in vivo, while also promoting the proliferation and activation of hepatic stellate cells (HSCs). Mechanistically, Sema3C interacted with NRP1 and ITGB1 in HSCs, activating downstream NF-kB signaling, thereby stimulating the release of IL-6 and upregulating HMGCR expression, consequently enhancing cholesterol synthesis in HSCs. Furthermore, CAF-secreted TGF-ß1 activates AP1 signaling to augment Sema3C expression in HCC cells, establishing a positive feedback loop that accelerates HCC progression. Notably, blockade of Sema3C effectively inhibits tumor growth and sensitizes HCC cells to sorafenib in vivo. In sum, our findings spotlight Sema3C as a novel biomarker facilitating the crosstalk between CSCs and stroma during hepatocarcinogenesis, thereby offering a promising avenue for enhancing treatment efficacy and overcoming drug resistance in HCC.

Asunto(s)

Carcinoma Hepatocelular , Neoplasias Hepáticas , Semaforinas , Microambiente Tumoral , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patología , Neoplasias Hepáticas/metabolismo , Humanos , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patología , Carcinoma Hepatocelular/metabolismo , Microambiente Tumoral/genética , Semaforinas/genética , Semaforinas/metabolismo , Integrina beta1/genética , Integrina beta1/metabolismo , Ratones , Transducción de Señal/genética , Células Estrelladas Hepáticas/metabolismo , Células Estrelladas Hepáticas/patología , Neuropilina-1/genética , Neuropilina-1/metabolismo , Línea Celular Tumoral , Células Madre Neoplásicas/patología , Células Madre Neoplásicas/metabolismo , Animales , Regulación Neoplásica de la Expresión Génica/genética , Sorafenib/farmacología , Fibroblastos Asociados al Cáncer/metabolismo , Fibroblastos Asociados al Cáncer/patología , Progresión de la Enfermedad

RESUMEN

The migration, proliferation, and apoptosis of trophoblastic cells play a crucial role in ensuring the effective preservation of pregnancy at the maternal-fetal interface. Any deviations in the structure and function of these cells might potentially result in the development of numerous pregnancy-related disorders, including missed abortion (MA). This study involved the examination of semaphorin 4A (SEMA4A) expression in missed abortion (n = 18) and normal early pregnancy (n = 18) villus. The findings of this study indicate a statistically significant decrease in the expression of SEMA4A in the villi of individuals diagnosed with missed abortion, as compared to the control group. The results of our vitro study showed that SEMA4A promoted the migration and proliferation of trophoblast cells and inhibited their apoptosis. Subsequent studies have shown that SEMA4A may be involved in regulating p-STAT3/STAT3, MMP9, bcl-2, and BAX levels. In summary, the findings of this study indicate a correlation between the decreased level of SEMA4A in chorionic villi and missed abortion. These results offer novel theoretical insights into the proper implantation and development of SEMA4A embryos at the maternal-fetal interface.

Asunto(s)

Apoptosis , Proliferación Celular , Factor de Transcripción STAT3 , Semaforinas , Transducción de Señal , Trofoblastos , Humanos , Femenino , Trofoblastos/metabolismo , Embarazo , Semaforinas/metabolismo , Semaforinas/genética , Factor de Transcripción STAT3/metabolismo , Adulto , Movimiento Celular , Vellosidades Coriónicas/metabolismo , Aborto Retenido/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo

RESUMEN

The precise assembly of a functional nervous system relies on axon guidance cues. Beyond engaging their cognate receptors and initiating signaling cascades that modulate cytoskeletal dynamics, guidance cues also bind components of the extracellular matrix, notably proteoglycans, yet the role and mechanisms of these interactions remain poorly understood. We found that Drosophila secreted semaphorins bind specifically to glycosaminoglycan (GAG) chains of proteoglycans, showing a preference based on the degree of sulfation. Structural analysis of Sema2b unveiled multiple GAG-binding sites positioned outside canonical plexin-binding site, with the highest affinity binding site located at the C-terminal tail, characterized by a lysine-rich helical arrangement that appears to be conserved across secreted semaphorins. In vivo studies revealed a crucial role of the Sema2b C-terminal tail in specifying the trajectory of olfactory receptor neurons. We propose that secreted semaphorins tether to the cell surface through interactions with GAG chains of proteoglycans, facilitating their presentation to cognate receptors on passing axons.

Asunto(s)

Orientación del Axón , Proteínas de Drosophila , Proteoglicanos , Semaforinas , Transducción de Señal , Animales , Semaforinas/metabolismo , Semaforinas/genética , Proteoglicanos/metabolismo , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , Axones/metabolismo , Drosophila melanogaster/metabolismo , Glicosaminoglicanos/metabolismo , Sitios de Unión , Unión Proteica , Neuronas Receptoras Olfatorias/metabolismo

RESUMEN

In animals undergoing metamorphosis, the appearance of the nervous system is coincidently transformed by the morphogenesis of neurons. Such morphogenic alterations are exemplified in three types of intrinsic neurons in the Drosophila memory center. In contrast to the well-characterized remodeling of γ neurons, the morphogenesis of α/ß and α'/ß' neurons has not been adequately explored. Here, we show that mamo, a BTB-zinc finger transcription factor that acts as a terminal selector for α'/ß' neurons, controls the formation of the correct axonal pattern of α'/ß' neurons. Intriguingly, specific Mamo isoforms are preferentially expressed in α'/ß' neurons to regulate the expression of axon guidance molecule Semaphorin-1a. This action directs proper axon guidance in α'/ß' neurons, which is also crucial for wiring of α'/ß' neurons with downstream neurons. Taken together, our results provide molecular insights into how neurons establish correct axonal patterns in circuitry assembly during adult memory center construction.

Asunto(s)

Orientación del Axón , Proteínas de Drosophila , Memoria , Isoformas de Proteínas , Semaforinas , Animales , Axones/metabolismo , Drosophila melanogaster/metabolismo , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , Regulación del Desarrollo de la Expresión Génica , Memoria/fisiología , Metamorfosis Biológica/fisiología , Neuronas/metabolismo , Isoformas de Proteínas/metabolismo , Isoformas de Proteínas/genética , Semaforinas/metabolismo , Semaforinas/genética , Factores de Transcripción/metabolismo , Factores de Transcripción/genética

RESUMEN

The anterior visceral endoderm (AVE) differs from the surrounding visceral endoderm (VE) in its migratory behavior and ability to restrict primitive streak formation to the opposite side of the mouse embryo. To characterize the molecular bases for the unique properties of the AVE, we combined single-cell RNA sequencing of the VE prior to and during AVE migration with phosphoproteomics, high-resolution live-imaging, and short-term lineage labeling and intervention. This identified the transient nature of the AVE with attenuation of "anteriorizing" gene expression as cells migrate and the emergence of heterogeneities in transcriptional states relative to the AVE's position. Using cell communication analysis, we identified the requirement of semaphorin signaling for normal AVE migration. Lattice light-sheet microscopy showed that Sema6D mutants have abnormalities in basal projections and migration speed. These findings point to a tight coupling between transcriptional state and position of the AVE and identify molecular controllers of AVE migration.

Asunto(s)

Movimiento Celular , Endodermo , Regulación del Desarrollo de la Expresión Génica , Animales , Endodermo/metabolismo , Endodermo/citología , Ratones , Transducción de Señal , Semaforinas/metabolismo , Semaforinas/genética , Embrión de Mamíferos/metabolismo , Embrión de Mamíferos/citología , Vísceras/metabolismo , Vísceras/embriología , Tipificación del Cuerpo/genética

RESUMEN

Semaphorin-plexin signaling plays a major role in the tumor microenvironment (TME). In particular, Semaphorin 4D (SEMA4D) has been shown to promote tumor growth and metastasis; however, the role of its high-affinity receptor Plexin-B1 (PLXNB1), which is expressed in the TME, is poorly understood. In this study, we directly targeted PLXNB1 in the TME of triple-negative murine breast carcinoma to elucidate its relevance in cancer progression. We found that primary tumor growth and metastatic dissemination were strongly reduced in PLXNB1-deficient mice, which showed longer survival. PLXNB1 loss in the TME induced a switch in the polarization of tumor-associated macrophages (TAM) toward a pro-inflammatory M1 phenotype and enhanced the infiltration of CD8+ T lymphocytes both in primary tumors and in distant metastases. Moreover, PLXNB1 deficiency promoted a shift in the Th1/Th2 balance of the T-cell population and an antitumor gene signature, with the upregulation of Icos, Perforin-1, Stat3, and Ccl5 in tumor-infiltrating lymphocytes (TILs). We thus tested the translational relevance of TME reprogramming driven by PLXNB1 inactivation for responsiveness to immunotherapy. Indeed, in the absence of PLXNB1, the efficacy of anti-PD-1 blockade was strongly enhanced, efficiently reducing tumor growth and distant metastasis. Consistent with this, pharmacological PLXNB1 blockade by systemic treatment with a specific inhibitor significantly hampered breast cancer growth and enhanced the antitumor activity of the anti-PD-1 treatment in a preclinical model. Altogether, these data indicate that PLXNB1 signaling controls the antitumor immune response in the TME and highlight this receptor as a promising immune therapeutic target for metastatic breast cancers.

Asunto(s)

Microambiente Tumoral , Animales , Microambiente Tumoral/inmunología , Femenino , Ratones , Humanos , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Línea Celular Tumoral , Neoplasias de la Mama/inmunología , Neoplasias de la Mama/patología , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/genética , Receptores de Superficie Celular/metabolismo , Receptores de Superficie Celular/genética , Macrófagos Asociados a Tumores/inmunología , Macrófagos Asociados a Tumores/metabolismo , Metástasis de la Neoplasia , Ratones Noqueados , Linfocitos Infiltrantes de Tumor/inmunología , Linfocitos Infiltrantes de Tumor/metabolismo , Neoplasias de la Mama Triple Negativas/inmunología , Neoplasias de la Mama Triple Negativas/patología , Neoplasias de la Mama Triple Negativas/metabolismo , Linfocitos T CD8-positivos/inmunología , Semaforinas/genética

RESUMEN

Purpose: The aim of this study was to elucidate the role of Sema4D in the pathogenesis of senescence-associated choroidal neovascularization (CNV) and to explore its underlying mechanisms. Methods: In this study, we utilized a model of laser-induced CNV in both young (3 months old) and old (18 months old) mice, including those with or without Sema4D knockout. The expression and localization of Sema4D in CNV were assessed using PCR, Western blot, and immunostaining. Subsequently, the morphological and imaging examinations were used to evaluate the size of CNV and vascular leakage. Finally, the expression of M2 markers, senescence-related markers, and molecules involved in the RhoA/ROCK pathway was detected. Results: We found that Sema4D was predominantly expressed in macrophages within CNV lesions, and both the mRNA and protein levels of Sema4D progressively increased following laser photocoagulation, a trend more pronounced in old mice. Moreover, Sema4D knockout markedly inhibited M2 polarization in senescent macrophages and reduced the size and leakage of CNV, particularly in aged mice. Mechanistically, aging was found to upregulate RhoA/ROCK signaling, and knockout of Sema4D effectively suppressed the activation of this pathway, with more significant effects observed in aged mice. Conclusions: Our findings revealed that the deletion of Sema4D markedly inhibited M2 macrophage polarization through the suppression of the RhoA/ROCK pathway, ultimately leading to the attenuation of senescence-associated CNV. These data indicate that targeting Sema4D could offer a promising approach for gene editing therapy in patients with neovascular age-related macular degeneration.

Asunto(s)

Neovascularización Coroidal , Modelos Animales de Enfermedad , Macrófagos , Ratones Endogámicos C57BL , Ratones Noqueados , Semaforinas , Transducción de Señal , Quinasas Asociadas a rho , Proteína de Unión al GTP rhoA , Animales , Neovascularización Coroidal/metabolismo , Neovascularización Coroidal/genética , Neovascularización Coroidal/patología , Ratones , Macrófagos/metabolismo , Quinasas Asociadas a rho/metabolismo , Semaforinas/genética , Semaforinas/metabolismo , Transducción de Señal/fisiología , Proteína de Unión al GTP rhoA/metabolismo , Antígenos CD/metabolismo , Antígenos CD/genética , Western Blotting , Masculino , Angiografía con Fluoresceína

RESUMEN

Accurate non-invasive biomarkers to diagnose metabolic dysfunction-associated steatotic liver disease (MASLD)-related fibrosis are urgently needed. This study applies a translational approach to develop a blood-based biomarker panel for fibrosis detection in MASLD. A molecular gene expression signature identified from a diet-induced MASLD mouse model (LDLr-/-.Leiden) is translated into human blood-based biomarkers based on liver biopsy transcriptomic profiles and protein levels in MASLD patient serum samples. The resulting biomarker panel consists of IGFBP7, SSc5D and Sema4D. LightGBM modeling using this panel demonstrates high accuracy in predicting MASLD fibrosis stage (F0/F1: AUC = 0.82; F2: AUC = 0.89; F3/F4: AUC = 0.87), which is replicated in an independent validation cohort. The overall accuracy of the model outperforms predictions by the existing markers Fib-4, APRI and FibroScan. In conclusion, here we show a disease mechanism-related blood-based biomarker panel with three biomarkers which is able to identify MASLD patients with mild or advanced hepatic fibrosis with high accuracy.

Asunto(s)

Biomarcadores , Cirrosis Hepática , Semaforinas , Humanos , Cirrosis Hepática/sangre , Cirrosis Hepática/diagnóstico , Cirrosis Hepática/patología , Biomarcadores/sangre , Animales , Masculino , Ratones , Femenino , Semaforinas/sangre , Semaforinas/genética , Semaforinas/metabolismo , Persona de Mediana Edad , Hígado Graso/sangre , Hígado Graso/diagnóstico , Hígado Graso/patología , Hígado/patología , Hígado/metabolismo , Modelos Animales de Enfermedad , Receptores de LDL/genética , Receptores de LDL/metabolismo , Transcriptoma , Ratones Noqueados , Adulto , Ratones Endogámicos C57BL , Proteínas de Unión a Factor de Crecimiento Similar a la Insulina

RESUMEN

Age-related diseases pose great challenges to health care systems worldwide. During aging, endothelial senescence increases the risk for cardiovascular disease. Recently, it was described that Phosphatase 1 Nuclear Targeting Subunit (PNUTS) has a central role in cardiomyocyte aging and homeostasis. Here, we determine the role of PNUTS in endothelial cell aging. We confirm that PNUTS is repressed in senescent endothelial cells (ECs). Moreover, PNUTS silencing elicits several of the hallmarks of endothelial aging: senescence, reduced angiogenesis and loss of barrier function. Findings are validate in vivo using endothelial-specific inducible PNUTS-deficient mice (Cdh5-CreERT2;PNUTSfl/fl), termed PNUTSEC-KO. Two weeks after PNUTS deletion, PNUTSEC-KO mice present severe multiorgan failure and vascular leakage. Transcriptomic analysis of PNUTS-silenced HUVECs and lungs of PNUTSEC-KO mice reveal that the PNUTS-PP1 axis tightly regulates the expression of semaphorin 3B (SEMA3B). Indeed, silencing of SEMA3B completely restores barrier function after PNUTS loss-of-function. These results reveal a pivotal role for PNUTS in endothelial homeostasis through a SEMA3B downstream pathway that provides a potential target against the effects of aging in ECs.

Asunto(s)

Senescencia Celular , Células Endoteliales de la Vena Umbilical Humana , Semaforinas , Animales , Humanos , Ratones , Envejecimiento/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/genética , Células Endoteliales/metabolismo , Endotelio Vascular/metabolismo , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Glicoproteínas de Membrana/metabolismo , Glicoproteínas de Membrana/genética , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/genética , Semaforinas/metabolismo , Semaforinas/genética

RESUMEN

AIMS: Bone morphogenetic protein-9 (BMP9) is critical for bone morphogenetic protein receptor type-2 (BMPR2) signalling in pulmonary vascular endothelial cells. Furthermore, human genetics studies support the central role of disrupted BMPR2 mediated BMP9 signalling in vascular endothelial cells in the initiation of pulmonary arterial hypertension (PAH). In addition, loss-of-function mutations in BMP9 have been identified in PAH patients. BMP9 is considered to play an important role in vascular homeostasis and quiescence. METHODS AND RESULTS: We identified a novel BMP9 target as the class-3 semaphorin, SEMA3G. Although originally identified as playing a role in neuronal development, class-3 semaphorins may have important roles in endothelial function. Here we show that BMP9 transcriptional regulation of SEMA3G occurs via ALK1 and the canonical Smad pathway, requiring both Smad1 and Smad5. Knockdown studies demonstrated redundancy between type-2 receptors in that BMPR2 and ACTR2A were compensatory. Increased SEMA3G expression by BMP9 was found to be regulated by the transcription factor, SOX17. Moreover, we observed that SEMA3G regulates VEGF signalling by inhibiting VEGFR2 phosphorylation and that VEGF, in contrast to BMP9, negatively regulated SEMA3G transcription. Functional endothelial cell assays of VEGF-mediated migration and network formation revealed that BMP9 inhibition of VEGF was abrogated by SEMA3G knockdown. Conversely, treatment with recombinant SEMA3G partially mimicked the inhibitory action of BMP9 in these assays. CONCLUSIONS: This study provides further evidence for the anti-angiogenic role of BMP9 in microvascular endothelial cells and these functions are mediated at least in part via SOX17 and SEMA3G induction.

Asunto(s)

Movimiento Celular , Células Endoteliales , Factor 2 de Diferenciación de Crecimiento , Semaforinas , Transducción de Señal , Factor A de Crecimiento Endotelial Vascular , Humanos , Movimiento Celular/efectos de los fármacos , Semaforinas/metabolismo , Semaforinas/genética , Factor 2 de Diferenciación de Crecimiento/genética , Factor 2 de Diferenciación de Crecimiento/metabolismo , Células Endoteliales/metabolismo , Células Endoteliales/efectos de los fármacos , Factor A de Crecimiento Endotelial Vascular/metabolismo , Factor A de Crecimiento Endotelial Vascular/genética , Proteína Smad5/metabolismo , Proteína Smad5/genética , Receptores de Activinas Tipo I/metabolismo , Receptores de Activinas Tipo I/genética , Receptores de Proteínas Morfogenéticas Óseas de Tipo II/metabolismo , Receptores de Proteínas Morfogenéticas Óseas de Tipo II/genética , Proteína Smad1/metabolismo , Proteína Smad1/genética , Pulmón/metabolismo , Pulmón/irrigación sanguínea , Neovascularización Fisiológica/efectos de los fármacos , Células Cultivadas

RESUMEN

Visual circuit development is characterized by subdivision of neuropils into layers that house distinct sets of synaptic connections. We find that, in the Drosophila medulla, this layered organization depends on the axon guidance regulator Plexin A. In Plexin A null mutants, synaptic layers of the medulla neuropil and arborizations of individual neurons are wider and less distinct than in controls. Analysis of semaphorin function indicates that Semaphorin 1a, acting in a subset of medulla neurons, is the primary partner for Plexin A in medulla lamination. Removal of the cytoplasmic domain of endogenous Plexin A has little effect on the formation of medulla layers; however, both null and cytoplasmic domain deletion mutations of Plexin A result in an altered overall shape of the medulla neuropil. These data suggest that Plexin A acts as a receptor to mediate morphogenesis of the medulla neuropil, and as a ligand for Semaphorin 1a to subdivide it into layers. Its two independent functions illustrate how a few guidance molecules can organize complex brain structures by each playing multiple roles.

Asunto(s)

Proteínas de Drosophila , Morfogénesis , Proteínas del Tejido Nervioso , Neurópilo , Lóbulo Óptico de Animales no Mamíferos , Receptores de Superficie Celular , Semaforinas , Animales , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , Semaforinas/metabolismo , Semaforinas/genética , Proteínas del Tejido Nervioso/metabolismo , Proteínas del Tejido Nervioso/genética , Morfogénesis/genética , Neurópilo/metabolismo , Lóbulo Óptico de Animales no Mamíferos/metabolismo , Lóbulo Óptico de Animales no Mamíferos/embriología , Receptores de Superficie Celular/metabolismo , Receptores de Superficie Celular/genética , Drosophila melanogaster/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/embriología , Neuronas/metabolismo , Drosophila/metabolismo , Drosophila/embriología , Mutación/genética

RESUMEN

The axons of retinal ganglion cells (RGCs) form the optic nerve, transmitting visual information from the eye to the brain. Damage or loss of RGCs and their axons is the leading cause of visual functional defects in traumatic injury and degenerative diseases such as glaucoma. However, there are no effective clinical treatments for nerve damage in these neurodegenerative diseases. Here, we report that LIM homeodomain transcription factor Lhx2 promotes RGC survival and axon regeneration in multiple animal models mimicking glaucoma disease. Furthermore, following N-methyl-D-aspartate (NMDA)-induced excitotoxicity damage of RGCs, Lhx2 mitigates the loss of visual signal transduction. Mechanistic analysis revealed that overexpression of Lhx2 supports axon regeneration by systematically regulating the transcription of regeneration-related genes and inhibiting transcription of Semaphorin 3C (Sema3C). Collectively, our studies identify a critical role of Lhx2 in promoting RGC survival and axon regeneration, providing a promising neural repair strategy for glaucomatous neurodegeneration.

Asunto(s)

Axones , Modelos Animales de Enfermedad , Glaucoma , Proteínas con Homeodominio LIM , Regeneración Nerviosa , Células Ganglionares de la Retina , Factores de Transcripción , Animales , Células Ganglionares de la Retina/metabolismo , Células Ganglionares de la Retina/patología , Proteínas con Homeodominio LIM/metabolismo , Proteínas con Homeodominio LIM/genética , Glaucoma/genética , Glaucoma/patología , Glaucoma/metabolismo , Factores de Transcripción/metabolismo , Factores de Transcripción/genética , Axones/metabolismo , Axones/patología , Ratones , Regeneración Nerviosa/genética , Regeneración Nerviosa/fisiología , Ratones Endogámicos C57BL , Supervivencia Celular/genética , Semaforinas/metabolismo , Semaforinas/genética , N-Metilaspartato/metabolismo