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In the present study, we examined the role of MDM2 in the angiogenesis process and its potential association with the sprouting of endothelial tip cells. To address this, we performed hypoxia-treated gastric cancer cells (HGC-27) to quantitative RT-PCR and Western blot analysis to measure the levels of MDM2 and VEGF-A mRNA and protein expression. Subsequently, we employed siRNA to disrupt MDM2 expression, followed by hypoxia treatment. The expression levels of MDM2 and VEGF-A mRNA and protein were subsequently reassessed. Additionally, ELISA was utilized to quantify the secretion levels of VEGF-A in each experimental group. A conditioned medium derived from HGC-27 cells treated with different agents was employed to assess its influence on the formation of EA.hy926 endothelial tip cells, using various techniques including Transwell plates migration assays, wound healing experiments, vascular formation assays, scanning electron microscopy, and immunofluorescence staining. These findings demonstrated that the in vitro knockdown of MDM2 in the conditioned medium exhibited significant inhibitory effects on endothelial cell migration, wound healing, and vascular formation. Additionally, the intervention led to a reduction in the presence of CD34+ tip cells and the formation of filopodia in endothelial cells, while partially restoring the integrity of tight junctions. Subsequent examination utilizing RNA-seq revealed that the suppression of MDM2 in HGC-27 cells resulted in the downregulation of the PI3K/AKT signaling pathway. Consequently, this downregulation led to an elevation in angiogenic effects induced by hypoxia.
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BACKGROUND: Muscle atrophy can cause muscle dysfunction and weakness. Krüppel-like factor 13 (KLF13), a central regulator of cellular energy metabolism, is highly expressed in skeletal muscles and implicated in the pathogenesis of several diseases. This study investigated the role of KLF13 in muscle atrophy, which could be a novel therapeutic target. METHODS: The effects of gene knockdown and pharmacological targeting of KLF13 on skeletal muscle atrophy were investigated using cell-based and animal models. Clofoctol, an antibiotic and KLF13 agonist, was also investigated as a candidate for repurposing. The mechanisms related to skeletal muscle atrophy were assessed by measuring the expression levels and activation statuses of key regulatory pathways and validated using gene knockdown and RNA sequencing. RESULTS: In a dexamethasone-induced muscle atrophy mouse model, the KLF13 knockout group had decreased muscle strength (N) (1.77 ± 0.10 vs. 1.48 ± 0.16, P < 0.01), muscle weight (%) [gastrocnemius (Gas): 76.0 ± 5.69 vs. 60.7 ± 7.23, P < 0.001; tibialis anterior (TA): 75.8 ± 6.21 vs. 67.5 ± 5.01, P < 0.05], and exhaustive running distance (m) (495.5 ± 64.8 vs. 315.5 ± 60.9, P < 0.05) compared with the control group. KLF13 overexpression preserved muscle mass (Gas: 100 ± 6.38 vs. 120 ± 14.4, P < 0.01) and the exhaustive running distance (423.8 ± 59.04 vs. 530.2 ± 77.45, P < 0.05) in an in vivo diabetes-induced skeletal muscle atrophy model. Clofoctol treatment protected against dexamethasone-induced muscle atrophy. Myotubes treated with dexamethasone, an atrophy-inducing glucocorticoid, were aggravated by KLF13 knockout, but anti-atrophic effects were achieved by inducing KLF13 overexpression. We performed a transcriptome analysis and luciferase reporter assays to further explore this mechanism, finding that delta-like 4 (Dll4) was a novel target gene of KLF13. The KLF13 transcript repressed Dll4, inhibiting the Dll4-Notch2 axis and preventing muscle atrophy. Dexamethasone inhibited KLF13 expression by inhibiting myogenic differentiation 1 (i.e., MYOD1)-mediated KLF13 transcriptional activation and promoting F-Box and WD repeat domain containing 7 (i.e., FBXW7)-mediated KLF13 ubiquitination. CONCLUSIONS: This study sheds new light on the mechanisms underlying skeletal muscle atrophy and potential drug targets. KLF13 regulates muscle atrophy and is a potential therapeutic target. Clofoctol is an attractive compound for repurposing studies to treat skeletal muscle atrophy.
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BACKGROUND: Angiogenesis and cancer metastasis depend on the DLL4/Notch signaling pathway. A new approach to treating angiogenesis could inhibit or block this pathway. In the present study, we investigated DLL4 expression as a biomarker capable of predicting survival outcomes in gastric cancer patients using a novel anti-DLL4 Nanobody. PATIENTS AND METHODS: By using a recently developed anti-DLL4 Nanobody, the expression of DLL4 was evaluated in tissue samples from 135 gastric cancer patients. It was evaluated whether DLL4 expression is related to clinicopathological factors, overall survival (OS), and recurrence-free survival (RFS). RESULTS: Sixty-five (48%) gastric cancer patients had a positive expression of DLL4 within the tumor tissue. Based on both the univariate and multivariate regression analyses, the expression of DLL4 was strongly associated with RFS (HR, 1.94; p = 0.008) and OS (HR, 2.06; p = 0.004). Moreover, the survival analysis demonstrated that DLL4 expression was a significant independent factor of unfavorable OS (HR, 2.7; p = 0.01) and RFS (HR, 2.3; p = 0.02) in gastric cancer patients. CONCLUSION: DLL4 expression in gastric cancer patients may predict poor prognosis and survival. Furthermore, the current data demonstrate the potential of Nanobody for detecting DLL4, and it may lead to develop novel therapies and diagnostics for tumors.
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Treatment options for patients with biliary tract cancer are limited, and the prognosis is poor. CTX-009, a novel bispecific antibody targeting both DLL4 and VEGF-A, has demonstrated antitumor activity in patients with advanced cancers as both a monotherapy and in combination with chemotherapy. In a phase II study of patients with advanced biliary tract cancer who had received one or two prior therapies, CTX-009 with paclitaxel demonstrated a 37.5% overall response rate (ORR). Described here is the design of and rationale for COMPANION-002, a randomized phase II/III study, which will evaluate the safety and efficacy of CTX-009 in combination with paclitaxel versus paclitaxel alone as second-line treatment for patients with advanced biliary tract cancer. The primary end point is ORR, and crossover is allowed.Clinical Trial Registration: NCT05506943 (ClinicalTrials.gov).
Looking for new options for patients with advanced biliary tract cancer? Explore COMPANION-002, Compass Therapeutics' phase II/III study of CTX-009 + paclitaxel as a second line treatment.#CMPX #biotech #healthcare #rarecancer.
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Pulmonary arterial hypertension (PAH) is a progressive cardiopulmonary disease characterized by vascular remodeling of small pulmonary arteries. Endothelial dysfunction in advanced PAH is associated with proliferation, apoptosis resistance, and endothelial to mesenchymal transition (EndoMT) due to aberrant signaling. DLL4, a cell membrane associated NOTCH ligand, activates NOTCH1 signaling and plays a pivotal role maintaining vascular integrity. Inhibition of DLL4 has been associated with the development of pulmonary hypertension, but the mechanism is incompletely understood. Here we report that BMPR2 silencing in PAECs activated AKT and decreased DLL4 expression. DLL4 loss was also seen in lungs of patients with IPAH and HPAH. Over-expression of DLL4 in PAECs induced BMPR2 promoter activity and exogenous DLL4 increased BMPR2 mRNA through NOTCH1 activation. Furthermore, DLL4/NOTCH1 signaling blocked AKT activation, decreased proliferation and reversed EndoMT in BMPR2-silenced PAECs and ECs from IPAH patients. PPARγ, suppressed by BMPR2 loss, was induced and activated by DLL4/NOTCH1 signaling in both BMPR2-silenced and IPAH PAECs, reversing aberrant phenotypic changes, in part through AKT inhibition. Finally, leniolisib, a well-tolerated oral PI3Kδ/AKT inhibitor, decreased cell proliferation, induced apoptosis and reversed markers of EndoMT in BMPR2-silenced PAECs. Restoring DLL4/NOTCH1/PPARγ signaling and/or suppressing AKT activation may be beneficial in preventing or reversing the pathologic vascular remodeling of PAH.
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BACKGROUND: Retinopathy of Prematurity (ROP) is a proliferative retinal vascular disease occurring in the retina of premature infants and is the main cause of childhood blindness. Nowadays anti-VEGF and retinal photocoagulation are mainstream treatments for ROP, but they develop a variety of complications. Hydrogen (H2) is widely considered as a useful neuroprotective and antioxidative therapeutic method for hypoxic-ischemic disease without toxic effects. However, whether H2 provides physiological angiogenesis promotion, neovascularization suppression and glial protection in the progression of ROP is largely unknown.This study aims to investigate the effects of H2 on retinal angiogenesis, neovascularization and neuroglial dysfunction in the retinas of oxygen-induced retinopathy (OIR) mice. METHODS: In this study, mice that were seven days old and either wild-type (WT) or Nrf2-deficient (Nrf2-/-) were exposed to 75% oxygen for 5 days and then returned to normal air conditions. Different stages of hydrogen gas (H2) inhalation were administered. Vascular obliteration, neovascularization, and blood vessel leakage were analyzed and compared. To count the number of neovascularization endothelial nuclei, routine HE staining of retinal sections was conducted. Immunohistochemistry was performed using DyLight 594 labeled GSL I-isolectin B4 (IB4), as well as primary antibodies against proliferating cell nuclear antigen (PCNA), glial fibrillary acidic protein (GFAP), and Iba-1. Western blots were used to measure the expression of NF-E2-related factor 2 (Nrf2), vascular endothelial growth factor (VEGF), Notch1, Dll4, and HIF-1α. Additionally, the expression of target genes such as NQO1, HO-1, Notch1, Hey1, Hey2, and Dll4 was measured. Human umbilical vein endothelial cells (HUVECs) treated with H2 under hypoxia were used as an in vitro model. RT-PCR was used to evaluate the mRNA expression of Nrf2, Notch/Dll4, and the target genes. The expression of reactive oxygen species (ROS) was observed using immunofluorescence staining. RESULTS: Our results indicate that 3-4% H2 does not disturb retinal physiological angiogenesis, but ameliorates vaso-obliteration and neovascularization in OIR mice. Moreover, H2 prevents the decreased density and reverses the morphologic and functional changes in retinal astrocytes caused by oxygen-induced injury. In addition, H2 inhalation reduces microglial activation, especially in the area of neovascularization in OIR mice. H2 plays a protective role in vascular regeneration by promoting Nrf2 activation and suppressing the Dll4-induced Notch signaling pathway in vivo. Also, H2 promotes the proliferation of HUVECs under hypoxia by negatively regulating the Dll4/Notch pathway and reducing ROS levels through Nrf2 pathway aligning with our findings in vivo.Moreover, the retinal oxygen-sensing mechanisms (HIF-1α/VEGF) are also involved in hydrogen-mediated retinal revascularization and neovascularization suppression. CONCLUSIONS: Collectively, our results indicate that H2 could be a promising therapeutic agent for POR treatment and that its beneficial effect in human ROP might involve the activation of the Nrf2-Notch axis as well as HIF-1α/VEGF pathways.
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Modelos Animales de Enfermedad , Hidrógeno , Neuroglía , Oxígeno , Neovascularización Retiniana , Retinopatía de la Prematuridad , Animales , Hidrógeno/farmacología , Neovascularización Retiniana/tratamiento farmacológico , Neuroglía/efectos de los fármacos , Ratones , Retinopatía de la Prematuridad/tratamiento farmacológico , Ratones Endogámicos C57BL , Retina/efectos de los fármacos , Animales Recién Nacidos , Regeneración/efectos de los fármacos , Inmunohistoquímica , Vasos Retinianos/efectos de los fármacosRESUMEN
NOTCH3 receptor signaling has been linked to the regulation of smooth muscle cell proliferation and the maintenance of smooth muscle cells in an undifferentiated state. Pulmonary arterial hypertension (World Health Organization Group 1 idiopathic disease: PAH) is a fatal disease characterized clinically by elevated pulmonary vascular resistance caused by extensive vascular smooth muscle cell proliferation, perivascular inflammation, and asymmetric neointimal hyperplasia in precapillary pulmonary arteries. In this review, a detailed overview of the specific role of NOTCH3 signaling in PAH, including its mechanisms of activation by a select ligand, downstream signaling effectors, and physiologic effects within the pulmonary vascular tree, is provided. Animal models showing the importance of the NOTCH3 pathway in clinical PAH will be discussed. New drugs and biologics that inhibit NOTCH3 signaling and reverse this deadly disease are highlighted.
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Hipertensión Arterial Pulmonar , Receptor Notch3 , Transducción de Señal , Humanos , Receptor Notch3/metabolismo , Receptor Notch3/genética , Animales , Hipertensión Arterial Pulmonar/metabolismo , Hipertensión Arterial Pulmonar/patología , Arteria Pulmonar/metabolismo , Arteria Pulmonar/patología , Hipertensión Pulmonar/metabolismo , Hipertensión Pulmonar/patología , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patología , Miocitos del Músculo Liso/metabolismo , Miocitos del Músculo Liso/patologíaRESUMEN
Pulmonary arterial hypertension (PAH) is a progressive cardiopulmonary disease characterized by pathologic vascular remodeling of small pulmonary arteries. Endothelial dysfunction in advanced PAH is associated with proliferation, apoptosis resistance, and endothelial to mesenchymal transition (EndoMT) due to aberrant signaling. DLL4, a cell membrane associated NOTCH ligand, plays a pivotal role maintaining vascular integrity. Inhibition of DLL4 has been associated with the development of pulmonary hypertension, but the mechanism is incompletely understood. Here we report that BMPR2 silencing in pulmonary artery endothelial cells (PAECs) activated AKT and suppressed the expression of DLL4. Consistent with these in vitro findings, increased AKT activation and reduced DLL4 expression was found in the small pulmonary arteries of patients with PAH. Increased NOTCH1 activation through exogenous DLL4 blocked AKT activation, decreased proliferation and reversed EndoMT. Exogenous and overexpression of DLL4 induced BMPR2 and PPRE promoter activity, and BMPR2 and PPARG mRNA in idiopathic PAH (IPAH) ECs. PPARγ, a nuclear receptor associated with EC homeostasis, suppressed by BMPR2 loss was induced and activated by DLL4/NOTCH1 signaling in both BMPR2-silenced and IPAH ECs, reversing aberrant phenotypic changes, in part through AKT inhibition. Directly blocking AKT or restoring DLL4/NOTCH1/PPARγ signaling may be beneficial in preventing or reversing the pathologic vascular remodeling of PAH.
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Receptores de Proteínas Morfogenéticas Óseas de Tipo II , Células Endoteliales , PPAR gamma , Proteínas Proto-Oncogénicas c-akt , Arteria Pulmonar , Receptor Notch1 , Transducción de Señal , Humanos , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptores de Proteínas Morfogenéticas Óseas de Tipo II/metabolismo , Receptores de Proteínas Morfogenéticas Óseas de Tipo II/genética , PPAR gamma/metabolismo , PPAR gamma/genética , Receptor Notch1/metabolismo , Receptor Notch1/genética , Arteria Pulmonar/metabolismo , Arteria Pulmonar/patología , Células Endoteliales/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas de Unión al Calcio/metabolismo , Proteínas de Unión al Calcio/genética , Hipertensión Arterial Pulmonar/metabolismo , Hipertensión Arterial Pulmonar/genética , Hipertensión Arterial Pulmonar/patología , Masculino , Proliferación Celular , Hipertensión Pulmonar/metabolismo , Hipertensión Pulmonar/genética , Hipertensión Pulmonar/patología , Femenino , Células CultivadasRESUMEN
Prolyl hydroxylase domain (PHD) proteins are oxygen sensors that use intracellular oxygen as a substrate to hydroxylate hypoxia-inducible factor (HIF) α proteins, routing them for polyubiquitylation and proteasomal degradation. Typically, HIFα accumulation in hypoxic or PHD-deficient tissues leads to upregulated angiogenesis. Here, we report unexpected retinal phenotypes associated with endothelial cell (EC)-specific gene targeting of Phd2 (Egln1) and Hif2alpha (Epas1). EC-specific Phd2 disruption suppressed retinal angiogenesis, despite HIFα accumulation and VEGFA upregulation. Suppressed retinal angiogenesis was observed both in development and in the oxygen-induced retinopathy (OIR) model. On the other hand, EC-specific deletion of Hif1alpha (Hif1a), Hif2alpha, or both did not affect retinal vascular morphogenesis. Strikingly, retinal angiogenesis appeared normal in mice double-deficient for endothelial PHD2 and HIF2α. In PHD2-deficient retinal vasculature, delta-like 4 (DLL4, a NOTCH ligand) and HEY2 (a NOTCH target) were upregulated by HIF2α-dependent mechanisms. Inhibition of NOTCH signaling by a chemical inhibitor or DLL4 antibody partially rescued retinal angiogenesis. Taken together, our data demonstrate that HIF2α accumulation in retinal ECs inhibits rather than stimulates retinal angiogenesis, in part by upregulating DLL4 expression and NOTCH signaling.
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Animales Recién Nacidos , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Células Endoteliales , Prolina Dioxigenasas del Factor Inducible por Hipoxia , Receptores Notch , Neovascularización Retiniana , Transducción de Señal , Regulación hacia Arriba , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Ratones , Receptores Notch/metabolismo , Receptores Notch/genética , Prolina Dioxigenasas del Factor Inducible por Hipoxia/metabolismo , Prolina Dioxigenasas del Factor Inducible por Hipoxia/genética , Neovascularización Retiniana/metabolismo , Neovascularización Retiniana/genética , Neovascularización Retiniana/patología , Células Endoteliales/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Retina/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Factor A de Crecimiento Endotelial Vascular/genética , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Proteínas de Unión al Calcio/metabolismo , Proteínas de Unión al Calcio/genética , Vasos Retinianos/metabolismo , AngiogénesisRESUMEN
The mechanisms governing brain vascularization during development remain poorly understood. A key regulator of developmental vascularization is delta like 4 (DLL4), a Notch ligand prominently expressed in endothelial cells (EC). Exposure to hyperoxia in premature infants can disrupt the development and functions of cerebral blood vessels and lead to long-term cognitive impairment. However, its role in cerebral vascular development and the impact of postnatal hyperoxia on DLL4 expression in mouse brain EC have not been explored. We determined the DLL4 expression pattern and its downstream signalling gene expression in brain EC using Dll4+/+ and Dll4+/LacZ mice. We also performed in vitro studies using human brain microvascular endothelial cells. Finally, we determined Dll4 and Cldn5 expression in mouse brain EC exposed to postnatal hyperoxia. DLL4 is expressed in various cell types, with EC being the predominant one in immature brains. Moreover, DLL4 deficiency leads to persistent abnormalities in brain microvasculature and increased vascular permeability both in vivo and in vitro. We have identified that DLL4 insufficiency compromises endothelial integrity through the NOTCH-NICD-RBPJ-CLDN5 pathway, resulting in the downregulation of the tight junction protein claudin 5 (CLDN5). Finally, exposure to neonatal hyperoxia reduces DLL4 and CLDN5 expression in developing mouse brain EC. We reveal that DLL4 is indispensable for brain vascular development and maintaining the blood-brain barrier's function and is repressed by neonatal hyperoxia. We speculate that reduced DLL4 signalling in brain EC may contribute to the impaired brain development observed in neonates exposed to hyperoxia. KEY POINTS: The role of delta like 4 (DLL4), a Notch ligand in vascular endothelial cells, in brain vascular development and functions remains unknown. We demonstrate that DLL4 is expressed at a high level during postnatal brain development in immature brains and DLL4 insufficiency leads to abnormal cerebral vasculature and increases vascular permeability both in vivo and in vitro. We identify that DLL4 regulates endothelial integrity through NOTCH-NICD-RBPJ-CLDN5 signalling. Dll4 and Cldn5 expression are decreased in mouse brain endothelial cells exposed to postnatal hyperoxia.
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Proteínas Adaptadoras Transductoras de Señales , Animales Recién Nacidos , Proteínas de Unión al Calcio , Claudina-5 , Células Endoteliales , Hiperoxia , Receptores Notch , Animales , Humanos , Ratones , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Encéfalo/metabolismo , Encéfalo/irrigación sanguínea , Encéfalo/crecimiento & desarrollo , Proteínas de Unión al Calcio/metabolismo , Proteínas de Unión al Calcio/genética , Células Cultivadas , Claudina-5/metabolismo , Claudina-5/genética , Células Endoteliales/metabolismo , Hiperoxia/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Péptidos y Proteínas de Señalización Intracelular/genética , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/genética , Ratones Endogámicos C57BL , Receptores Notch/metabolismo , Receptores Notch/genética , Transducción de SeñalRESUMEN
The tumour microenvironment (TME) drives bladder cancer (BLCA) progression. Targeting the TME has emerged as a promising strategy for BLCA treatment in recent years. Furthermore, checkpoint blockade therapies are only beneficial for a minority of patients with BLCA, and drug resistance is a barrier to achieving significant clinical effects of anti-programmed cell death protein-1 (PD-1)/programmed death protein ligand-1 (PD-L1) therapy. In this study, higher low-density lipoprotein receptor-related protein 1 (LRP1) levels were related to a poorer prognosis for patients with various cancers, including those with higher grades and later stages of BLCA. Enrichment analysis demonstrated that LRP1 plays a role in the epithelial-mesenchymal transition (EMT), NOTCH signalling pathway, and ubiquitination. LRP1 knockdown in BLCA cells delayed BLCA progression both in vivo and in vitro. Furthermore, LRP1 knockdown suppressed EMT, reduced DLL4-NOTCH2 signalling activity, and downregulated M2-like macrophage polarisation. Patients with BLCA and higher LRP1 levels responded weakly to anti-PD-1 therapy in the IMvigor210 cohort. Moreover, LRP1 knockdown enhanced the therapeutic effects of anti-PD-1 in mice. Taken together, our findings suggest that LRP1 is a potential target for improving the efficacy of anti-PD-1/PD-L1 therapy by preventing EMT and M2-like macrophage polarisation by blocking the DLL4-NOTCH2 axis.
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Resistencia a Antineoplásicos , Transición Epitelial-Mesenquimal , Proteína 1 Relacionada con Receptor de Lipoproteína de Baja Densidad , Receptor Notch2 , Transducción de Señal , Microambiente Tumoral , Neoplasias de la Vejiga Urinaria , Humanos , Neoplasias de la Vejiga Urinaria/tratamiento farmacológico , Neoplasias de la Vejiga Urinaria/patología , Neoplasias de la Vejiga Urinaria/metabolismo , Neoplasias de la Vejiga Urinaria/genética , Neoplasias de la Vejiga Urinaria/inmunología , Proteína 1 Relacionada con Receptor de Lipoproteína de Baja Densidad/metabolismo , Proteína 1 Relacionada con Receptor de Lipoproteína de Baja Densidad/genética , Animales , Ratones , Transición Epitelial-Mesenquimal/efectos de los fármacos , Línea Celular Tumoral , Receptor Notch2/metabolismo , Receptor Notch2/genética , Macrófagos/metabolismo , Macrófagos/inmunología , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Receptor de Muerte Celular Programada 1/metabolismo , Receptor de Muerte Celular Programada 1/antagonistas & inhibidores , Receptor de Muerte Celular Programada 1/genética , Proteínas de Unión al Calcio/metabolismo , Proteínas de Unión al Calcio/genética , Inhibidores de Puntos de Control Inmunológico/farmacología , Inhibidores de Puntos de Control Inmunológico/uso terapéutico , Femenino , Masculino , Ensayos Antitumor por Modelo de Xenoinjerto , Quimiocina CCL2RESUMEN
Background: Currently, bone marrow mesenchymal stem cells (BMSCs) have been reported to promote endometrial regeneration in rat models of mechanically injury-induced uterine adhesions (IUAs), but the therapeutic effects and mechanisms of hypoxic BMSC-derived exosomes on IUAs have not been elucidated. Objective: To investigate the potential mechanism by which the BMSCS-derived exosomal miR-424-5p regulates IUA angiogenesis through the DLL4/Notch signaling pathway under hypoxic conditions and promotes endometrial injury repair. Methods: The morphology of the exosomes was observed via transmission electron microscopy, and the expression of exosome markers (CD9, CD63, CD81, and HSP70) was detected via flow cytometry and Western blotting. The expression of angiogenesis-related genes (Ang1, Flk1, Vash1, and TSP1) was detected via RTâqPCR, and the expression of DLL4/Notch signaling pathway-related proteins (DLL4, Notch1, and Notch2) was detected via Western blotting. Cell proliferation was detected by a CCK-8 assay, and angiogenesis was assessed via an angiogenesis assay. The expression of CD3 was detected by immunofluorescence. The endometrial lesions of IUA rats were observed via HE staining, and the expression of CD3 and VEGFA was detected via immunohistochemistry. Results: Compared with those in exosomes from normoxic conditions, miR-424-5p was more highly expressed in the exosomes from hypoxic BMSCs. Compared with those in normoxic BMSC-derived exosomes, the proliferation and angiogenesis of HUVECs were significantly enhanced after treatment with hypoxic BMSC-derived exosomes, and these effects were weakened after inhibition of miR-424-5p. miR-424-5p can target and negatively regulate the expression of DLL4, promote the expression of the proangiogenic genes Ang1 and Flk1, and inhibit the expression of the antiangiogenic genes Vash1 and TSP1. The effect of miR-424-5p can be reversed by overexpression of DLL4. In IUA rats, treatment with hypoxic BMSC exosomes and the miR-424-5p mimic promoted angiogenesis and improved endometrial damage. Conclusion: The hypoxic BMSC-derived exosomal miR-424-5p promoted angiogenesis and improved endometrial injury repair by regulating the DLL4/Notch signaling pathway, which provides a new idea for the treatment of IUAs.
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Exosomas , Células Madre Mesenquimatosas , MicroARNs , Enfermedades Uterinas , Animales , Femenino , Ratas , Proteínas Adaptadoras Transductoras de Señales/genética , Angiogénesis , Proteínas de Unión al Calcio/genética , Exosomas/genética , MicroARNs/genética , Transducción de Señal/genética , Enfermedades Uterinas/metabolismoRESUMEN
INTRODUCTION: Angiogenesis is fundamental for tumor growth and metastasis across many solid malignancies. Considerable interest has focused on the molecular regulation of tumor angiogenesis as a means to predict disease outcomes and guide therapeutic decisions. METHODS: In the present study, we investigated the prognostic value of transforming growth factor beta (TGF-ß), epidermal growth factor (EGF), fibroblast growth factor (FGF), delta-like ligand 4 (DLL4), and vascular endothelial growth factor (VEGF) in the serum of 120 women diagnosed with breast cancer using ELISA as well as examined their associations with clinical parameters and the outcome of the disease. RESULTS: Our results demonstrated that the serum concentration of TGF-ß and EGF were remarkably higher in patients with higher tumor size, end stages of the disease, and positive lymph node involvement compared to patients with lower tumor size, early stages of the disease, and negative lymph node involvement. In addition, we found a significant correlation between the serum concentration of VEGF and the level of EGF, FGF, and DLL4 in patients with breast cancer. Furthermore, both univariate and multivariate analyses showed that TGF-ß and EGF can be used as end-stage predictors. DISCUSSION/CONCLUSION: Based on our findings, increasing the level of angiogenesis factors is significantly associated with higher tumor size and late stages of the disease in patients with breast cancer. Moreover, measuring the level of angiogenesis factors could lead to better prediction of disease outcomes and choosing the best treatments for patients.
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Neoplasias de la Mama , Humanos , Femenino , Neoplasias de la Mama/diagnóstico , Neoplasias de la Mama/metabolismo , Factor A de Crecimiento Endotelial Vascular , Factor de Crecimiento Epidérmico , Pronóstico , Angiogénesis , Factores de Crecimiento Endotelial Vascular , Factor de Crecimiento Transformador beta/metabolismo , Biomarcadores de Tumor/metabolismoRESUMEN
Notch and its ligands play a critical role in rheumatoid arthritis (RA) pathogenesis. Hence, studies were conducted to delineate the functional significance of the Notch pathway in RA synovial tissue (ST) cells and the influence of RA therapies on their expression. Morphological studies reveal that JAG1, DLL4, and Notch1 are highly enriched in RA ST lining and sublining CD68+CD14+ MΦs. JAG1 and DLL4 transcription is jointly upregulated in RA MΦs reprogrammed by TLR4/5 ligation and TNF, whereas Syntenin-1 exposure expands JAG1, DLL4, and Notch1 expression levels in these cells. Single-cell RNA-seq data exhibit that JAG1 and Notch3 are overexpressed on all fibroblast-like synoviocyte (FLS) subpopulations, in parallel, JAG2, DLL1, and Notch1 expression levels are modest on RA FLS and are predominately potentiated by TLR4 ligation. Intriguingly, JAG1, DLL1/4, and Notch1/3 are presented on RA endothelial cells, and their expression is mutually reconfigured by TLR4/5 ligation in the endothelium. Synovial JAG1/JAG2/DLL1 or Notch1/3 transcriptomes were unchanged in patients who received disease-modifying anti-rheumatic drugs (DMARDs) or IL-6R Ab therapy regardless of disease activity score. Uniquely, RA MΦs and endothelial cells rewired by IL-6 displayed DLL4 transcriptional upregulation, and IL-6R antibody treatment disrupted RA ST DLL4 transcription in good responders compared to non-responders or moderate responders. Nevertheless, the JAG1/JAG2/DLL1/DLL4 transcriptome was diminished in anti-TNF good responders with myeloid pathotype and was unaltered in the fibroid pathotype except for DLL4. Taken together, our findings suggest that RA myeloid Notch ligands can serve as markers for anti-TNF responsiveness and trans-activate Notch receptors expressed on RA FLS and/or endothelial cells.
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Artritis Reumatoide , Inhibidores del Factor de Necrosis Tumoral , Humanos , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Proteínas de Unión al Calcio/metabolismo , Proteínas de la Membrana/metabolismo , Proteína Jagged-1/genética , Proteína Jagged-1/metabolismo , Células Endoteliales/metabolismo , Receptor Toll-Like 4/metabolismo , Receptores Notch/metabolismo , Biomarcadores , Artritis Reumatoide/tratamiento farmacológico , Ligandos , Receptor Notch1/metabolismoRESUMEN
PURPOSE: Vascular endothelium plays a central role in the pathogenesis of acute and chronic radiation injuries, yet the mechanisms which promote sustained endothelial dysfunction and contribute to late responding organ failure are unclear. We employed 2nd window (> 1100 nm emission) Near-Infrared (NIR) imaging using indocyanine green (ICG) to track and define the role of the notch ligand Delta-like ligand 4 (Dll4) in mediating vascular injury in two late-responding radiosensitive organs: the lung and kidney. PROCEDURES: Consomic strains of female Salt Sensitive or SS (Dll4-high) and SS with 3rd chromosome inherited from Brown Norway, SS.BN3 (Dll4-low) rats at ages 11-12 weeks were used to demonstrate the impact of reduced Dll4 expression on long-term vascular integrity, renal function, and survival following high-dose 13 Gy partial body irradiation at 42- and 90 days post-radiation. 2nd window dynamic NIR fluorescence imaging with ICG was analyzed with physiology-based pharmacokinetic modeling and confirmed with assays of endothelial Dll4 expression to assess the role of endogenous Dll4 expression on radiation injury protection. RESULTS: We show that SS.BN3 (Dll4-low) rats are relatively protected from vascular permeability disruption compared to the SS (Dll4-high) strain. We further demonstrated that SS.BN3 (Dll4-low) rats have reduced radiation induced loss of CD31+ vascular endothelial cells, and increased Dll4 vascular expression is correlated with vascular dysfunction. CONCLUSIONS: Together, these data suggest Dll4 plays a key role in pathogenesis of radiation-induced vascular injury to the lung and kidney.
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Proteínas de la Membrana , Traumatismos por Radiación , Lesiones del Sistema Vascular , Ratas , Femenino , Animales , Células Endoteliales/metabolismo , Lesiones del Sistema Vascular/diagnóstico por imagen , Lesiones del Sistema Vascular/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismoRESUMEN
BACKGROUND: Blocking Delta-like 4 (DLL4)/Notch has emerged as a promising therapeutic target for the treatment of tumours by deregulating angiogenesis. However, DLL4/Notch serves as a negative regulator of angiogenesis in multiple organs while acting as a positive regulator of H-type angiogenesis in postnatal long bones. Therefore, the effect of DLL4/Notch signalling blockade on mandibular condylar osteogenesis attracted our attention. OBJECTIVE: To explore the effect of blocking DLL4/Notch on mandibular advancement (MA)-induced condylar osteogenesis. METHODS: Six-week-old young male C57BL/6J mice (n = 40) were randomly divided into four groups: control group, MA group, MA + Anti-DLL4 group and MA + IgG group. Of note, IgG served as the isotype control for the anti-DLL4. The femurs, tibias and mandibular condyles were collected after sacrificing mice on Day 31 for morphology, micro-computed tomography, immunofluorescence, histology and immunohistochemistry evaluation. RESULTS: First, DLL4/Notch blockade shortened femoral length and reduced bone mass by inhibiting H-type angiogenesis. Second, DLL4/Notch blockade disrupted MA-induced condylar head volume and quality by inhibiting H-type angiogenesis. Mechanistically, blocking DLL4/Notch reduced the number of runt-related transcription factor 2+ (RUNX2+ ) early osteoprogenitors and the expression of Noggin protein in the condylar subchondral bone by inhibiting H-type angiogenesis. In addition, blockade of DLL4/Notch also destroyed the condylar cartilage layer. CONCLUSION: DLL4/Notch blockade results in shortened femurs and osteopenia, as well as impaired MA-induced condylar osteogenic volume and quality in growing mice by inhibiting H-type angiogenesis. Therefore, when blocking DLL4/Notch is used as a treatment target for diseases, attention should be paid to its impact on the bone mass of mandibular condyle.
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Avance Mandibular , Osteogénesis , Masculino , Animales , Ratones , Ratones Endogámicos C57BL , Cóndilo Mandibular , Angiogénesis , Microtomografía por Rayos X , Inmunoglobulina GRESUMEN
Background: Anti-angiogenic pathways are important for inhibiting tumor growth and migration. Tryptanthrin has anticancer properties in vivo but its anti-angiogenesis activities and associated mechanisms remain unclear. Methods: The effects of tryptanthrin were investigated in vivo using fluorescent labeling of blood vessels in zebrafish. Fluorescence quantitation was conducted to analyze the level of delta-like ligand 4 (Dll4) gene expression. Transcriptome sequencing and quantitative polymerase chain reaction (qPCR) analyses were performed to explore the molecular mechanisms of anti-tumor angiogenesis. Results: Significant anti-tumor effects were observed in all 48-hpf (hours post-fertilization) zebrafish treated with tryptanthrin (P<0.05). The 6-hpf zebrafish were cultured to 48 and 72 hpf following tryptanthrin treatment. It was found that compared with the control groups, the fluorescence area and the number of complete internode vessels reduced significantly following treatment with medium and high concentrations of tryptanthrin (P<0.05). The relative expression of Dll4 in the 48-hpf zebrafish was significantly inhibited only in the high concentration group (P<0.05). qPCR analysis revealed that the levels of Krt18b, desma, Tnnt2c, and Krt4 gene expression were significantly up-regulated in zebrafish following Dll4 overexpression. After Dll4 knockdown, the level of desma and Tnnt2c gene expression was significantly up-regulated. Conclusions: Tryptanthrin can inhibit tumor growth in vivo in a concentration-dependent manner by down-regulating Dll4 protein expression, and at the same time up-regulating the level of desma and Tnnt2c gene expression.
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Suppressing tumors through anti-angiogenesis has been established as an effective clinical treatment strategy. Bevacizumab, a monoclonal antibody, is commonly used in various indications. However, two major challenges limit the long-term efficacy of bevacizumab: drug resistance and side effects. Bevacizumab resistance has been extensively studied at the molecular level, but no drug candidates have been developed for clinical use to overcome this resistance. In a previous study conducted by our team, a major finding was that high expression of ESM1 in bevacizumab-resistant tumors is associated with an unfavorable response to treatment. In particular, an increase in ESM1 expression contributes to heightened lung metastasis and microvascular density, which ultimately decreases the tumor's sensitivity to bevacizumab. In contrast, the silencing of ESM1 results in reduced angiogenesis and suppressed tumor growth in tumors resistant to bevacizumab. We put forward the hypothesis that targeting ESM1 could serve as a therapeutic strategy in overcoming bevacizumab resistance. In this study, a variety of anti-ESM1 antibodies with high affinity to human ESM1 were successfully prepared and characterized. Our in vivo study confirmed the establishment of a bevacizumab-resistant human colorectal cancer model and further demonstrated that the addition of anti-ESM1 monoclonal antibodies to bevacizumab treatment significantly improved tumor response while downregulating DLL4 and MMP9. In conclusion, our study suggests that anti-hESM1 monoclonal antibodies have the potential to alleviate or overcome bevacizumab resistance, thereby providing new strategies and drug candidates for clinical research in the treatment of bevacizumab-resistant colorectal cancer.
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Neoplasias Colorrectales , Neoplasias Pulmonares , Humanos , Bevacizumab/farmacología , Bevacizumab/uso terapéutico , Anticuerpos Monoclonales Humanizados/uso terapéutico , Factor A de Crecimiento Endotelial Vascular , Anticuerpos Monoclonales/farmacología , Anticuerpos Monoclonales/uso terapéutico , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/patología , Inhibidores de la Angiogénesis/farmacología , Inhibidores de la Angiogénesis/uso terapéutico , Neovascularización Patológica/patología , Proteínas de Neoplasias , ProteoglicanosRESUMEN
Tubular epithelial cells (TECs) play critical roles in the development of diabetic nephropathy (DN), and can activate macrophages through the secretion of exosomes. However, the mechanism(s) of TEC-exosomes in macrophage activation under DN remains unknown. By mass spectrometry, 1,644 differentially expressed proteins, especially Dll4, were detected in the urine exosomes of DN patients compared with controls, which was confirmed by western blot assay. Elevated Epsin1 and Dll4/N1ICD expression was observed in kidney tissues in both DN patients and db/db mice and was positively associated with tubulointerstitial damage. Exosomes from high glucose (HG)-treated tubular cells (HK-2) with Epsin1 knockdown (KD) ameliorated macrophage activation, TNF-α, and IL-6 expression, and tubulointerstitial damage in C57BL/6 mice in vivo. In an in vitro study, enriched Dll4 was confirmed in HK-2 cells stimulated with HG, which was captured by THP-1 cells and promoted M1 macrophage activation. In addition, Epsin1 modulated the content of Dll4 in TEC-exosomes stimulated with HG. TEC-exosomes with Epsin1-KD significantly inhibited N1ICD activation and iNOS expression in THP-1 cells compared with incubation with HG alone. These findings suggested that Epsin1 could modulate tubular-macrophage crosstalk in DN by mediating exosomal sorting of Dll4 and Notch1 activation.
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Diabetes Mellitus , Nefropatías Diabéticas , Animales , Ratones , Movimiento Celular , Diabetes Mellitus/metabolismo , Nefropatías Diabéticas/metabolismo , Células Epiteliales/metabolismo , Glucosa/metabolismo , Macrófagos/metabolismo , Ratones Endogámicos C57BLRESUMEN
Delta like canonical notch ligand 4 (Dll4) expression levels in tumors are known to affect the efficacy of cancer therapies. This study aimed to develop a model to predict Dll4 expression levels in tumors using dynamic enhanced near-infrared (NIR) imaging with indocyanine green (ICG). Two rat-based consomic xenograft (CXM) strains of breast cancer with different Dll4 expression levels and eight congenic xenograft strains were studied. Principal component analysis (PCA) was used to visualize and segment tumors, and modified PCA techniques identified and analyzed tumor and normal regions of interest (ROIs). The average NIR intensity for each ROI was calculated from pixel brightness at each time interval, yielding easily interpretable features including the slope of initial ICG uptake, time to peak perfusion, and rate of ICG intensity change after reaching half-maximum intensity. Machine learning algorithms were applied to select discriminative features for classification, and model performance was evaluated with a confusion matrix, receiver operating characteristic curve, and area under the curve. The selected machine learning methods accurately identified host Dll4 expression alterations with sensitivity and specificity above 90%. This may enable stratification of patients for Dll4 targeted therapies. NIR imaging with ICG can noninvasively assess Dll4 expression levels in tumors and aid in effective decision making for cancer therapy.