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While local nanoparticle delivery to lymph nodes is well studied, there are few design criteria for intravenous delivery to the entire lymph node repertoire. In this study, we investigated the effect of NP pH transition on lymph node targeting by employing a series of ultra-pH-sensitive (UPS) polymeric micelles. The UPS library responds to pH thresholds (pKa 6.9, 6.2, and 5.3) over a range of physiological pH. We observed a dependence of intravenous lymph node targeting on micelle pH transition. UPS6.9 (subscript indicates pKa) shows poor lymph node delivery, while UPS5.3 delivers efficiently to lymph node sets. We investigated targeting mechanisms of UPS5.3, observing an accumulation among lymph node lymphatics and a dependence on lymph node-resident macrophages. To overcome the pH-threshold barrier, which limits UPS6.9, we rationally designed a nanoparticle coassembly of UPS6.9 with UPS5.3, called HyUPS. The HyUPS micelle retains the constitutive pH transitions of each polymer, showing stepwise responses to discrete pH thresholds. We demonstrate that HyUPS improves UPS6.9 delivery to lymph nodes, extending this platform for disease detection of lymph node metastasis.
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Ganglios Linfáticos , Micelas , Concentración de Iones de Hidrógeno , Ganglios Linfáticos/metabolismo , Animales , Ratones , Nanopartículas/química , Polímeros/química , Femenino , Sistemas de Liberación de MedicamentosRESUMEN
Heterotopic ossification (HO) is a challenging condition that occurs after musculoskeletal injury and is characterized by the formation of bone in non-skeletal tissues. While the effect of HO on blood vessels is well established, little is known about its impact on lymphatic vessels. Here, we use a mouse model of traumatic HO to investigate the relationship between HO and lymphatic vessels. We show that injury triggers lymphangiogenesis at the injury site, which is associated with elevated vascular endothelial growth factor C (VEGF-C) levels. Through single-cell transcriptomic analyses, we identify mesenchymal progenitor cells and tenocytes as sources of Vegfc. We demonstrate by lineage tracing that Vegfc-expressing cells undergo osteochondral differentiation and contribute to the formation of HO. Last, we show that Vegfc haploinsufficiency results in a nearly 50% reduction in lymphangiogenesis and HO formation. These findings shed light on the complex mechanisms underlying HO formation and its impact on lymphatic vessels.
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Linfangiogénesis , Células Madre Mesenquimatosas , Osificación Heterotópica , Factor C de Crecimiento Endotelial Vascular , Animales , Osificación Heterotópica/metabolismo , Osificación Heterotópica/patología , Osificación Heterotópica/genética , Factor C de Crecimiento Endotelial Vascular/metabolismo , Factor C de Crecimiento Endotelial Vascular/genética , Ratones , Células Madre Mesenquimatosas/metabolismo , Vasos Linfáticos/metabolismo , Vasos Linfáticos/patología , Diferenciación Celular , Tenocitos/metabolismo , Osteogénesis , Haploinsuficiencia , Ratones Endogámicos C57BL , Modelos Animales de Enfermedad , MasculinoRESUMEN
Complex lymphatic anomalies (CLAs) are sporadically occurring diseases caused by the maldevelopment of lymphatic vessels. We and others recently reported that somatic activating mutations in KRAS can cause CLAs. However, the mechanisms by which activating KRAS mutations cause CLAs are poorly understood. Here, we show that KRASG12D expression in lymphatic endothelial cells (LECs) during embryonic development impairs the formation of lymphovenous valves and causes the enlargement of lymphatic vessels. We demonstrate that KRASG12D expression in primary human LECs induces cell spindling, proliferation, and migration. It also increases AKT and ERK1/2 phosphorylation and decreases the expression of genes that regulate the maturation of lymphatic vessels. We show that MEK1/2 inhibition with the FDA-approved drug trametinib suppresses KRASG12D-induced morphological changes, proliferation, and migration. Trametinib also decreases ERK1/2 phosphorylation and increases the expression of genes that regulate the maturation of lymphatic vessels. We also show that trametinib and Cre-mediated expression of a dominant-negative form of MEK1 (Map2k1 K97M) suppresses KRASG12D-induced lymphatic vessel hyperplasia in embryos. Last, we demonstrate that conditional knockout of wild-type Kras in LECs does not affect the formation or function of lymphatic vessels. Together, our data indicate that KRAS/MAPK signaling must be tightly regulated during embryonic development for the proper development of lymphatic vessels and further support the testing of MEK1/2 inhibitors for treating CLAs.
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Objective: Gorham-Stout disease (GSD) is a rare lymphatic anomaly that can be caused by somatic activating mutations in KRAS. This discovery has led investigators to suggest that MEK inhibitors could be a novel treatment for GSD. However, the effect of MEK inhibitors on bone disease in animal models of GSD has not been investigated. We recently reported that Osx-tTA;TetO-Vegfc mice exhibit a phenotype that resembles GSD. Osx-tTA;TetO-Vegfc mice overexpress VEGF-C in bone, which stimulates the development of lymphatic vessels in bone and the gradual loss of cortical bone. The objective of this study was to characterize the effect of trametinib, an FDA-approved MEK1/2 inhibitor, on lymphangiogenesis and osteolysis in Osx-tTA;TetO-Vegfc mice. Methods: Immunoblotting was performed to assess the effect of trametinib on VEGF-C-induced phosphorylation of ERK1/2, AKT, and S6 in primary human lymphatic endothelial cells (LECs). Prevention and intervention experiments were performed to determine the effect of trametinib on lymphangiogenesis and osteolysis in Osx-tTA;TetO-Vegfc mice. Results: We found that trametinib blocked VEGF-C-induced phosphorylation of ERK1/2 in primary human LECs. We also found that trametinib prevented VEGF-C-induced lymphatic invasion of bone and cortical bone loss in Osx-tTA;TetO-Vegfc mice. Additionally, trametinib slowed the progression of disease in Osx-tTA;TetO-Vegfc mice with established disease. However, it did not reverse disease in Osx-tTA;TetO-Vegfc mice. Conclusion: Our results show trametinib impacts bone disease in Osx-tTA;TetO-Vegfc mice. These findings further support the testing of MEK inhibitors in patients with GSD and other RAS pathway-driven complex lymphatic anomalies with bone involvement.
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BACKGROUND: Lymphatic abnormalities are observed in several types of kidney disease, but the relationship between the renal lymphatic system and renal function is unclear. The discovery of lymphatic-specific proteins, advances in microscopy, and available genetic mouse models provide the tools to help elucidate the role of renal lymphatics in physiology and disease. METHODS: We utilized a mouse model containing a missense mutation in Vegfr3 (dubbed Chy ) that abrogates its kinase ability. Vegfr3 Chy/+ mice were examined for developmental abnormalities and kidney-specific outcomes. Control and Vegfr3 Chy/+ mice were subjected to cisplatin-mediated injury. We characterized renal lymphatics using tissue-clearing, light-sheet microscopy, and computational analyses. RESULTS: In the kidney, VEGFR3 is expressed not only in lymphatic vessels but also, in various blood capillaries. Vegfr3 Chy/+ mice had severely reduced renal lymphatics with 100% penetrance, but we found no abnormalities in BP, serum creatinine, BUN, albuminuria, and histology. There was no difference in the degree of renal injury after low-dose cisplatin (5 mg/kg), although Vegfr3 Chy/+ mice developed perivascular inflammation. Cisplatin-treated controls had no difference in total cortical lymphatic volume and length but showed increased lymphatic density due to decreased cortical volume. CONCLUSIONS: We demonstrate that VEGFR3 is required for development of renal lymphatics. Our studies reveal that reduced lymphatic density does not impair renal function at baseline and induces only modest histologic changes after mild injury. We introduce a novel quantification method to evaluate renal lymphatics in 3D and demonstrate that accurate measurement of lymphatic density in CKD requires assessment of changes to cortical volume.
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Cisplatino , Vasos Linfáticos , Ratones , Animales , Sistema Linfático/fisiología , Riñón/fisiología , Mutación , LinfangiogénesisRESUMEN
Gorham-Stout disease (GSD) is a sporadically occurring lymphatic disorder. Patients with GSD develop ectopic lymphatics in bone, gradually lose bone, and can have life-threatening complications, such as chylothorax. The etiology of GSD is poorly understood, and current treatments for this disease are inadequate for most patients. To explore the pathogenesis of GSD, we performed targeted high-throughput sequencing with samples from a patient with GSD and identified an activating somatic mutation in KRAS (p.G12V). To characterize the effect of hyperactive KRAS signaling on lymphatic development, we expressed an active form of KRAS (p.G12D) in murine lymphatics (iLECKras mice). We found that iLECKras mice developed lymphatics in bone, which is a hallmark of GSD. We also found that lymphatic valve development and maintenance was altered in iLECKras mice. Because most iLECKras mice developed chylothorax and died before they had significant bone disease, we analyzed the effect of trametinib (an FDA-approved MEK1/2 inhibitor) on lymphatic valve regression in iLECKras mice. Notably, we found that trametinib suppressed this phenotype in iLECKras mice. Together, our results demonstrate that somatic activating mutations in KRAS can be associated with GSD and reveal that hyperactive KRAS signaling stimulates the formation of lymphatics in bone and impairs the development of lymphatic valves. These findings provide insight into the pathogenesis of GSD and suggest that trametinib could be an effective treatment for GSD.
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Huesos/patología , Vasos Linfáticos , Osteólisis Esencial , Proteínas Proto-Oncogénicas p21(ras)/genética , Piridonas/farmacología , Pirimidinonas/farmacología , Acrilonitrilo/análogos & derivados , Acrilonitrilo/farmacología , Compuestos de Anilina/farmacología , Animales , Modelos Animales de Enfermedad , Mutación con Ganancia de Función , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Humanos , Linfangiogénesis/genética , Vasos Linfáticos/anomalías , Vasos Linfáticos/patología , Ratones , Osteólisis Esencial/genética , Osteólisis Esencial/patología , Transducción de Señal , Estructuras Linfoides Terciarias/genética , Estructuras Linfoides Terciarias/patologíaRESUMEN
Epithelioid hemangioendothelioma (EHE) is a poorly understood and devastating vascular cancer. Sequencing of EHE has revealed a unique gene fusion between the Hippo pathway nuclear effector TAZ (WWTR1) and the brain-enriched transcription factor CAMTA1 in â¼90% of cases. However, it remains unclear whether the TAZ-CAMTA1 gene fusion is a driver of EHE, and potential targeted therapies are unknown. Here, we show that TAZ-CAMTA1 expression in endothelial cells is sufficient to drive the formation of vascular tumors with the distinctive features of EHE, and inhibition of TAZ-CAMTA1 results in the regression of these vascular tumors. We further show that activated TAZ resembles TAZ-CAMTA1 in driving the formation of EHE-like vascular tumors, suggesting that constitutive activation of TAZ underlies the pathological features of EHE. We show that TAZ-CAMTA1 initiates an angiogenic and regenerative-like transcriptional program in endothelial cells, and disruption of the TAZ-CAMTA1-TEAD interaction or ectopic expression of a dominant negative TEAD in vivo inhibits TAZ-CAMTA1-mediated transformation. Our study provides the first genetic model of a TAZ fusion oncoprotein driving its associated human cancer, pinpointing TAZ-CAMTA1 as the key driver and a valid therapeutic target of EHE.
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Proteínas de Unión al Calcio/metabolismo , Carcinogénesis/genética , Células Endoteliales/patología , Regulación Neoplásica de la Expresión Génica , Hemangioendotelioma Epitelioide/genética , Hemangioendotelioma Epitelioide/patología , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Transactivadores/metabolismo , Animales , Proteínas de Unión al Calcio/genética , Línea Celular Tumoral , Fusión Génica , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Ratones , Transactivadores/genética , Proteínas Coactivadoras Transcripcionales con Motivo de Unión a PDZRESUMEN
Sporadic lymphatic diseases are orphans among orphans in the medical community, a diverse collection of disorders at the intersection of cardiac, gastrointestinal, pulmonary, dermatologic, and oncologic disease that receives only passing attention in medical school and that no subspecialty in medicine fully embraces as its own. They often present in a confusing and illusive manner, with a fractured bone, expectoration of blood or a branching airway cast, a swollen limb or a collection of chylous material; protean manifestations that can challenge even the most expert diagnostician. Yet many of these acquired disorders have been discovered to have a targetable genetic basis, and as the case report of Foster et al (2020) demonstrates, the sedulous clinician-patient dyad can be rewarded with an almost miraculous result when the molecular pathogenesis of the disease is pursued and an exquisitely targeted therapy is administered.
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Enfermedades Linfáticas , Vasos Linfáticos , HumanosRESUMEN
Bones do not normally have lymphatics. However, individuals with generalized lymphatic anomaly (GLA) or Gorham-Stout disease (GSD) develop ectopic lymphatics in bone. Despite growing interest in the development of tissue-specific lymphatics, the cellular origin of bone lymphatic endothelial cells (bLECs) is not known and the development of bone lymphatics has not been fully characterized. Here, we describe the development of bone lymphatics in mouse models of GLA and GSD. Through lineage-tracing experiments, we show that bLECs arise from pre-existing Prox1-positive LECs. We show that bone lymphatics develop in a stepwise manner where regional lymphatics grow, breach the periosteum and then invade bone. We also show that the development of bone lymphatics is impaired in mice that lack osteoclasts. Last, we show that rapamycin can suppress the growth of bone lymphatics in our models of GLA and GSD. In summary, we show that bLECs can arise from pre-existing LECs and that rapamycin can prevent the growth of bone lymphatics.
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Huesos/embriología , Vasos Linfáticos/embriología , Animales , Huesos/efectos de los fármacos , Linaje de la Célula/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Proteínas de Homeodominio/metabolismo , Humanos , Integrasas/metabolismo , Vasos Linfáticos/efectos de los fármacos , Ratones Transgénicos , Mutación/genética , Osteoclastos/efectos de los fármacos , Osteoclastos/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Sirolimus/farmacología , Factor de Transcripción Sp7/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
The discovery of meningeal lymphatic vessels (LVs) has sparked interest in identifying their role in diseases of the central nervous system. Similar to peripheral LVs, meningeal LVs depend on vascular endothelial growth factor receptor-3 (VEGFR3) signaling for development. Here we characterize the effect of stroke on meningeal LVs, and the impact of meningeal lymphatic hypoplasia on post-stroke outcomes. We show that photothrombosis (PT), but not transient middle cerebral artery occlusion (tMCAo), induces meningeal lymphangiogenesis in young male C57Bl/J6 mice. We also show that Vegfr3wt/mut mice develop significantly fewer meningeal LVs than Vegfr3wt/wt mice. Again, meningeal lymphangiogenesis occurs in the alymphatic zone lateral to the sagittal sinus only after PT-induced stroke in Vegfr3wt/wt mice. Interestingly, Vegfr3wt/mut mice develop larger stroke volumes than Vegfr3wt/wt mice after tMCAo, but not after PT. Our results reveal differences between PT and tMCAo models of stroke and underscore the need to consider method of stroke induction when investigating the role of meningeal lymphatics. Taken together, our data indicate that ischemic injury can induce the growth of meningeal LVs and that the absence of these LVs can impact post-stroke outcomes.
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Sistema Glinfático , Linfangiogénesis/genética , Accidente Cerebrovascular , Receptor 3 de Factores de Crecimiento Endotelial Vascular , Animales , Modelos Animales de Enfermedad , Sistema Glinfático/metabolismo , Sistema Glinfático/fisiopatología , Masculino , Ratones , Ratones Transgénicos , Accidente Cerebrovascular/genética , Accidente Cerebrovascular/metabolismo , Accidente Cerebrovascular/patología , Accidente Cerebrovascular/fisiopatología , Receptor 3 de Factores de Crecimiento Endotelial Vascular/genética , Receptor 3 de Factores de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Generalized lymphatic anomaly (GLA) is a vascular disorder characterized by diffuse or multifocal lymphatic malformations (LMs). The etiology of GLA is poorly understood. We identified four distinct somatic PIK3CA variants (Glu542Lys, Gln546Lys, His1047Arg, and His1047Leu) in tissue samples from five out of nine patients with GLA. These same PIK3CA variants occur in PIK3CA-related overgrowth spectrum and cause hyperactivation of the PI3K-AKT-mTOR pathway. We found that the mTOR inhibitor, rapamycin, prevented lymphatic hyperplasia and dysfunction in mice that expressed an active form of PIK3CA (His1047Arg) in their lymphatics. We also found that rapamycin reduced pain in patients with GLA. In conclusion, we report that somatic activating PIK3CA mutations can cause GLA, and we provide preclinical and clinical evidence to support the use of rapamycin for the treatment of this disabling and deadly disease.
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Fosfatidilinositol 3-Quinasa Clase I , Linfangioleiomiomatosis , Sistema Linfático , Mutación Missense , Sirolimus/administración & dosificación , Adolescente , Adulto , Sustitución de Aminoácidos , Niño , Preescolar , Fosfatidilinositol 3-Quinasa Clase I/genética , Fosfatidilinositol 3-Quinasa Clase I/metabolismo , Femenino , Humanos , Linfangioleiomiomatosis/diagnóstico por imagen , Linfangioleiomiomatosis/tratamiento farmacológico , Linfangioleiomiomatosis/enzimología , Linfangioleiomiomatosis/genética , Sistema Linfático/anomalías , Sistema Linfático/diagnóstico por imagen , Sistema Linfático/enzimología , Masculino , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Serina-Treonina Quinasas TOR/genética , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
Resistance to standard therapy remains a major challenge in the treatment of pancreatic ductal adenocarcinoma (PDA). Although anti-VEGF therapy delays PDA progression, therapy-induced hypoxia results in a less differentiated mesenchymal-like tumor cell phenotype, which reinforces the need for effective companion therapies. COX-2 inhibition has been shown to promote tumor cell differentiation and improve standard therapy response in PDA. Here, we evaluate the efficacy of COX-2 inhibition and VEGF blockade in preclinical models of PDA. In vivo, the combination therapy was more effective in limiting tumor growth and metastasis than single-agent therapy. Combination therapy also reversed anti-VEGF-induced epithelial-mesenchymal transition and collagen deposition and altered the immune landscape by increasing tumor-associated CD8+ T cells while reducing FoxP3+ T cells and FasL expression on the tumor endothelium. IMPLICATIONS: Together, these findings demonstrate that COX-2 inhibition enhances the efficacy of anti-VEGF therapy by reducing hypoxia-induced epithelial plasticity and promoting an immune landscape that might facilitate immune activation.Visual Overview: http://mcr.aacrjournals.org/content/molcanres/17/2/348/F1.large.jpg.
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Inhibidores de la Ciclooxigenasa 2/metabolismo , Neoplasias Pancreáticas/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Animales , Línea Celular Tumoral , Modelos Animales de Enfermedad , Femenino , Humanos , Ratones , Neoplasias Pancreáticas/patología , Microambiente TumoralRESUMEN
Ever since it was discovered that endothelial cells line lymphatic vessels, investigators have been working on unraveling the mechanisms that control the growth of this distinctive endothelium and its role in normal physiology and human disease. Recent technological advances have ushered in a new era of "omics" research on the lymphatic system. Research on the genome, transcriptome, proteome, and metabolome of lymphatics has increased our understanding of the biology of the lymphatic vasculature. Here, we introduce the context-lymphatic "systemomics," then briefly review some of the latest advances in research on tumor-associated lymphatic vessels highlighting several "omic" studies that have shed light on mechanisms controlling the growth and function of tumor-associated lymphatic vessels. We conclude by returning, with unanswered questions, to the larger context of cancer and the lymphatic system as a vasculature, circulation, route of entry and transport, and control center of the immune network.
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Linfangiogénesis/genética , Metástasis Linfática/genética , Vasos Linfáticos/metabolismo , Neoplasias/genética , Células Endoteliales , Genoma/genética , Humanos , Vasos Linfáticos/patología , Metaboloma/genética , Neoplasias/patología , Proteoma/genética , Transcriptoma/genéticaRESUMEN
Patients with Gorham-Stout disease (GSD) have lymphatic vessels in their bones and their bones gradually disappear. Here, we report that mice that overexpress VEGF-C in bone exhibit a phenotype that resembles GSD. To drive VEGF-C expression in bone, we generated Osx-tTA;TetO-Vegfc double-transgenic mice. In contrast to Osx-tTA mice, Osx-tTA;TetO-Vegfc mice developed lymphatics in their bones. We found that inhibition of VEGFR3, but not VEGFR2, prevented the formation of bone lymphatics in Osx-tTA;TetO-Vegfc mice. Radiological and histological analysis revealed that bones from Osx-tTA;TetO-Vegfc mice were more porous and had more osteoclasts than bones from Osx-tTA mice. Importantly, we found that bone loss in Osx-tTA;TetO-Vegfc mice could be attenuated by an osteoclast inhibitor. We also discovered that the mutant phenotype of Osx-tTA;TetO-Vegfc mice could be reversed by inhibiting the expression of VEGF-C. Taken together, our results indicate that expression of VEGF-C in bone is sufficient to induce the pathologic hallmarks of GSD in mice.
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Resorción Ósea/patología , Huesos/patología , Endotelio Linfático/patología , Vasos Linfáticos/patología , Osteoclastos/patología , Factor C de Crecimiento Endotelial Vascular/fisiología , Animales , Resorción Ósea/metabolismo , Huesos/metabolismo , Células Cultivadas , Endotelio Linfático/metabolismo , Humanos , Vasos Linfáticos/metabolismo , Ratones , Ratones Transgénicos , Osteoclastos/metabolismo , Fenotipo , Transducción de Señal , Receptor 3 de Factores de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Organ homeostasis relies upon cellular and molecular processes that restore tissue structure and function in a timely fashion. Lymphatic vessels help maintain fluid equilibrium by returning interstitial fluid that evades venous uptake back to the circulation. Despite its important role in tissue homeostasis, cancer metastasis, and close developmental origins with the blood vasculature, the number of molecular players known to control lymphatic system development is relatively low. Here we show, using genetic approaches in zebrafish and mice, that the endothelial specific microRNA mir-126, previously implicated in vascular integrity, regulates lymphatic development. In zebrafish, in contrast to mir-126 morphants, double mutants (mir-126a-/-; mir-126b-/-, hereafter mir-126-/-) do not exhibit defects in vascular integrity but develop lymphatic hypoplasia; mir-126-/- animals fail to develop complete trunk and facial lymphatics, display severe edema and die as larvae. Notably, following MIR-126 inhibition, human Lymphatic Endothelial Cells (hLECs) respond poorly to VEGFA and VEGFC. In this context, we identify a concomitant reduction in Vascular Endothelial Growth Factor Receptor-2 (VEGFR2) and Vascular Endothelial Growth Factor Receptor-3 (VEGFR3, also known as FLT4) expression upon MIR-126 inhibition. In vivo, we further show that flt4+/- zebrafish embryos exhibit lymphatic defects after mild miR-126 knockdown. Similarly, loss of Mir-126 in Flt4+/- mice results in embryonic edema and lethality. Thus, our results indicate that miR-126 modulation of Vegfr signaling is essential for lymphatic system development in fish and mammals.
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Linfangiogénesis/genética , MicroARNs/genética , Animales , Animales Modificados Genéticamente , Western Blotting , Técnicas de Cultivo de Célula , Células Endoteliales/metabolismo , Técnicas de Genotipaje , Humanos , Vasos Linfáticos/embriología , Vasos Linfáticos/metabolismo , Ratones , Transducción de Señal/genética , Factor A de Crecimiento Endotelial Vascular/metabolismo , Pez Cebra , Proteínas de Pez Cebra/genéticaRESUMEN
Gorham-Stout disease (GSD) is a rare bone disorder characterized by aggressive osteolysis associated with lymphatic vessel invasion within bone marrow cavities. The etiology of GSD is not known, and there is no effective therapy or animal model for the disease. Here, we investigated if lymphatic endothelial cells (LECs) affect osteoclasts (OCs) to cause a GSD osteolytic phenotype in mice. We examined the effect of a mouse LEC line on osteoclastogenesis in co-cultures. LECs significantly increased receptor activator of NF-κB ligand (RANKL)-mediated OC formation and bone resorption. LECs expressed high levels of macrophage colony-stimulating factor (M-CSF), but not RANKL, interleukin-6 (IL-6), and tumor necrosis factor (TNF). LEC-mediated OC formation and bone resorption were blocked by an M-CSF neutralizing antibody or Ki20227, an inhibitor of the M-CSF receptor, c-Fms. We injected LECs into the tibias of wild-type (WT) mice and observed massive osteolysis on X-ray and micro-CT scans. Histology showed that LEC-injected tibias had significant trabecular and cortical bone loss and increased OC numbers. M-CSF protein levels were significantly higher in serum and bone marrow plasma of mice given intra-tibial LEC injections. Immunofluorescence staining showed extensive replacement of bone and marrow by podoplanin+ LECs. Treatment of LEC-injected mice with Ki20227 significantly decreased tibial bone destruction. In addition, lymphatic vessels in a GSD bone sample were stained positively for M-CSF. Thus, LECs cause bone destruction in vivo in mice by secreting M-CSF, which promotes OC formation and activation. Blocking M-CSF signaling may represent a new therapeutic approach for treatment of patients with GSD. Furthermore, tibial injection of LECs is a useful mouse model to study GSD. © 2017 American Society for Bone and Mineral Research.
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Células Endoteliales/metabolismo , Factor Estimulante de Colonias de Macrófagos/sangre , Osteoclastos/metabolismo , Osteólisis Esencial/sangre , Animales , Línea Celular , Células Endoteliales/patología , Células Endoteliales/trasplante , Interleucina-6/metabolismo , Ratones , Osteoclastos/patología , Ligando RANK/metabolismo , Tibia/metabolismo , Tibia/patologíaRESUMEN
Lymphatic vessels modulate tissue fluid balance and inflammation and provide a conduit for endocrine and lipid transport. The growth of new lymphatic vessels in the adult, lymphangiogenesis, is predominantly mediated through vascular endothelial growth factor receptor-3 (VEGFR-3) signaling. We took advantage of the unique binding of murine VEGF-D specifically to VEGFR-3 and generated mice capable of inducible, tissue-specific expression of murine VEGF-D under a tightly-controlled tetracycline response element (TRE) promoter to stimulate adult tissue lymphangiogenesis. With doxycycline-activated expression, TRE-VEGF-D mouse crossed to mice with tissue-specific promoters for the lung [Clara cell secretory protein-reverse tetracycline transactivator (rtTA)] developed pulmonary lymphangiectasia. In the kidney, (kidney-specific protein-rtTA × TRE-VEGF-D) mice exhibited rapid lymphatic hyperplasia on induction of VEGF-D expression. Crossed with adipocyte-specific adiponectin-rtTA mice [Adipo-VEGF-D (VD)], chronic VEGF-D overexpression was capable of inducing de novo lymphangiogenesis in white adipose tissue and a massive expansion of brown adipose tissue lymphatics. VEGF-D expression in white adipose tissue also increased macrophage infiltration and tissue fibrosis in the tissue. Expression did not, however, measurably affect peripheral fluid transport, the blood vasculature, or basal metabolic parameters. On removal of the doxycycline stimulus, VEGF-D expression returned to normal, and the expanded adipose tissue lymphatics regressed in Adipo-VD mice. The inducible TRE-VEGF-D mouse thus provides a novel murine platform to study the adult mechanisms and therapies of an array of disease- and tissue-specific models of lymphangiogenesis.
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Tejido Adiposo Pardo/metabolismo , Tejido Adiposo Blanco/metabolismo , Riñón/metabolismo , Pulmón/metabolismo , Ganglios Linfáticos/metabolismo , Linfangiogénesis/genética , Vasos Linfáticos/patología , Factor D de Crecimiento Endotelial Vascular/genética , Tejido Adiposo Pardo/patología , Tejido Adiposo Blanco/patología , Animales , Composición Corporal , Femenino , Técnica del Anticuerpo Fluorescente , Hiperplasia , Riñón/patología , Pulmón/patología , Ganglios Linfáticos/patología , Masculino , Ratones , Ratones Transgénicos , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Receptor 3 de Factores de Crecimiento Endotelial VascularRESUMEN
It is well established that lung tumors induce the formation of lymphatic vessels. However, the molecular mechanisms controlling tumor lymphangiogenesis in lung cancer have not been fully delineated. In the present study, we identify a panel of non-small cell lung cancer (NSCLC) cell lines that induce lymphangiogenesis and use genome-wide mRNA expression to characterize the molecular mechanisms regulating tumor lymphangiogenesis. We show that Calu-1, H1993, HCC461, HCC827, and H2122 NSCLC cell lines form tumors that induce lymphangiogenesis whereas Calu-3, H1155, H1975, and H2073 NSCLC cell lines form tumors that do not induce lymphangiogenesis. By analyzing genome-wide mRNA expression data, we identify a 17-gene expression signature that distinguishes lymphangiogenic from non-lymphangiogenic NSCLC cell lines. Importantly, VEGF-C is the only lymphatic growth factor in this expression signature and is approximately 50-fold higher in the lymphangiogenic group than in the non-lymphangiogenic group. We show that forced expression of VEGF-C by H1975 cells induces lymphangiogenesis and that knockdown of VEGF-C in H1993 cells inhibits lymphangiogenesis. Additionally, we demonstrate that the triple angiokinase inhibitor, nintedanib (small molecule that blocks all FGFRs, PDGFRs, and VEGFRs), suppresses tumor lymphangiogenesis in H1993 tumors. Together, these data suggest that VEGF-C is the dominant driver of tumor lymphangiogenesis in NSCLC and reveal a specific therapy that could potentially block tumor lymphangiogenesis in NSCLC patients.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas/patología , Neoplasias Pulmonares/patología , Linfangiogénesis/genética , Transcriptoma , Animales , Línea Celular Tumoral , Variaciones en el Número de Copia de ADN/genética , Genómica , Humanos , Indoles/farmacología , Linfangiogénesis/efectos de los fármacos , Transcriptoma/efectos de los fármacos , Factor C de Crecimiento Endotelial Vascular/genéticaRESUMEN
Repurposing "old" drugs can facilitate rapid clinical translation but necessitates novel mechanistic insight. Warfarin, a vitamin K "antagonist" used clinically for the prevention of thrombosis for more than 50 years, has been shown to have anticancer effects. We hypothesized that the molecular mechanism underlying its antitumor activity is unrelated to its effect on coagulation, but is due to inhibition of the Axl receptor tyrosine kinase on tumor cells. Activation of Axl by its ligand Gas6, a vitamin K-dependent protein, is inhibited at doses of warfarin that do not affect coagulation. Here, we show that inhibiting Gas6-dependent Axl activation with low-dose warfarin, or with other tumor-specific Axl-targeting agents, blocks the progression and spread of pancreatic cancer. Warfarin also inhibited Axl-dependent tumor cell migration, invasiveness, and proliferation while increasing apoptosis and sensitivity to chemotherapy. We conclude that Gas6-induced Axl signaling is a critical driver of pancreatic cancer progression and its inhibition with low-dose warfarin or other Axl-targeting agents may improve outcome in patients with Axl-expressing tumors.