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AIMS: Circulating levels of sphingosine 1-phosphate (S1P), an HDL-associated ligand for endothelial cell (EC) protective S1P receptor-1 (S1PR1), are reduced in disease states associated with endothelial dysfunction. Yet as S1PR1 has high affinity for S1P and can be activated by ligand-independent mechanisms and EC-autonomous S1P production, it is unclear if relative reductions in circulating S1P impact endothelial function. It is also unclear how EC S1PR1 insufficiency, whether induced by ligand deficiency or by S1PR1-directed immunosuppressive therapy, affects different vascular subsets. METHODS AND RESULTS: We here fine-map the zonation of S1PR1 signalling in the murine blood and lymphatic vasculature, superimpose cell type-specific and relative deficiencies in S1P production to define ligand source- and dose-dependence, and correlate receptor engagement to essential functions. In naïve blood vessels, despite broad expression, EC S1PR1 engagement was restricted to resistance-size arteries, lung capillaries and high-endothelial venules (HEV). Similar zonation was observed for albumin extravasation in EC S1PR1 deficient mice, and brain extravasation was reproduced with arterial EC-selective S1pr1 deletion. In lymphatic EC, S1PR1 engagement was high in collecting vessels and lymph nodes and low in terminal capillaries that drain tissue fluids. While EC S1P production sustained S1PR1 signaling in lymphatics and HEV, hematopoietic cells provided â¼90% of plasma S1P and sustained signaling in resistance arteries and lung capillaries. S1PR1 signaling and endothelial function were both surprisingly sensitive to reductions in plasma S1P with apparent saturation around 50% of normal levels. S1PR1 engagement did not depend on sex or age, but modestly increased in arteries in hypertension and diabetes. Sphingosine kinase (Sphk)-2 deficiency also increased S1PR1 engagement selectively in arteries, which could be attributed to Sphk1-dependent S1P release from perivascular macrophages. CONCLUSIONS: This study highlights vessel subtype-specific S1PR1 functions and mechanisms of engagement and supports the relevance of S1P as circulating biomarker for endothelial function.
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Meningeal lymphatic vessels (MLVs) promote tissue clearance and immune surveillance in the central nervous system (CNS). Vascular endothelial growth factor-C (VEGF-C) regulates MLV development and maintenance and has therapeutic potential for treating neurological disorders. Herein, we investigated the effects of VEGF-C overexpression on brain fluid drainage and ischemic stroke outcomes in mice. Intracerebrospinal administration of an adeno-associated virus expressing mouse full-length VEGF-C (AAV-mVEGF-C) increased CSF drainage to the deep cervical lymph nodes (dCLNs) by enhancing lymphatic growth and upregulated neuroprotective signaling pathways identified by single nuclei RNA sequencing of brain cells. In a mouse model of ischemic stroke, AAV-mVEGF-C pretreatment reduced stroke injury and ameliorated motor performances in the subacute stage, associated with mitigated microglia-mediated inflammation and increased BDNF signaling in brain cells. Neuroprotective effects of VEGF-C were lost upon cauterization of the dCLN afferent lymphatics and not mimicked by acute post-stroke VEGF-C injection. We conclude that VEGF-C prophylaxis promotes multiple vascular, immune, and neural responses that culminate in a protection against neurological damage in acute ischemic stroke.
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Accidente Cerebrovascular Isquémico , Accidente Cerebrovascular , Animales , Ratones , Factor C de Crecimiento Endotelial Vascular , Enfermedades Neuroinflamatorias , DrenajeRESUMEN
The eye, an anatomical extension of the central nervous system (CNS), exhibits many molecular and cellular parallels to the brain. Emerging research demonstrates that changes in the brain are often reflected in the eye, particularly in the retina1. Still, the possibility of an immunological nexus between the posterior eye and the rest of the CNS tissues remains unexplored. Here, studying immune responses to herpes simplex virus in the brain, we observed that intravitreal immunization protects mice against intracranial viral challenge. This protection extended to bacteria and even tumours, allowing therapeutic immune responses against glioblastoma through intravitreal immunization. We further show that the anterior and posterior compartments of the eye have distinct lymphatic drainage systems, with the latter draining to the deep cervical lymph nodes through lymphatic vasculature in the optic nerve sheath. This posterior lymphatic drainage, like that of meningeal lymphatics, could be modulated by the lymphatic stimulator VEGFC. Conversely, we show that inhibition of lymphatic signalling on the optic nerve could overcome a major limitation in gene therapy by diminishing the immune response to adeno-associated virus and ensuring continued efficacy after multiple doses. These results reveal a shared lymphatic circuit able to mount a unified immune response between the posterior eye and the brain, highlighting an understudied immunological feature of the eye and opening up the potential for new therapeutic strategies in ocular and CNS diseases.
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Encéfalo , Ojo , Sistema Linfático , Animales , Femenino , Humanos , Masculino , Ratones , Conejos , Bacterias/inmunología , Encéfalo/anatomía & histología , Encéfalo/inmunología , Dependovirus/inmunología , Ojo/anatomía & histología , Ojo/inmunología , Glioblastoma/inmunología , Herpesvirus Humano 2/inmunología , Inyecciones Intravítreas , Sistema Linfático/anatomía & histología , Sistema Linfático/inmunología , Vasos Linfáticos/anatomía & histología , Vasos Linfáticos/inmunología , Macaca mulatta , Meninges/inmunología , Nervio Óptico/inmunología , Porcinos , Pez Cebra , Factor C de Crecimiento Endotelial Vascular/inmunología , Factor C de Crecimiento Endotelial Vascular/metabolismo , Factor C de Crecimiento Endotelial Vascular/farmacologíaRESUMEN
Meningeal lymphatic vessels promote tissue clearance and immune surveillance in the central nervous system (CNS). Vascular endothelium growth factor-C (VEGF-C) is essential for meningeal lymphatic development and maintenance and has therapeutic potential for treating neurological disorders, including ischemic stroke. We have investigated the effects of VEGF-C overexpression on brain fluid drainage, single cell transcriptome in the brain, and stroke outcomes in adult mice. Intra-cerebrospinal fluid administration of an adeno-associated virus expressing VEGF-C (AAV-VEGF-C) increases the CNS lymphatic network. Post-contrast T1 mapping of the head and neck showed that deep cervical lymph node size and drainage of CNS-derived fluids were increased. Single nuclei RNA sequencing revealed a neuro-supportive role of VEGF-C via upregulation of calcium and brain-derived neurotrophic factor (BDNF) signaling pathways in brain cells. In a mouse model of ischemic stroke, AAV-VEGF-C pretreatment reduced stroke injury and ameliorated motor performances in the subacute stage. AAV-VEGF-C thus promotes CNS-derived fluid and solute drainage, confers neuroprotection, and reduces ischemic stroke damage. Short abstract: Intrathecal delivery of VEGF-C increases the lymphatic drainage of brain-derived fluids confers neuroprotection, and improves neurological outcomes after ischemic stroke.
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Background: The blood brain barrier (BBB) preserves neuronal function in the central nervous system (CNS) by tightly controlling metabolite exchanges with the blood. In the eye, the retina is likewise protected by the blood-retina barrier (BRB) to maintain phototransduction. We showed that the secreted guidance cue Netrin-1 regulated BBB integrity, by binding to endothelial Unc5B and regulating canonical ß-catenin dependent expression of BBB gene expression. Objective: Here, we investigated if Netrin-1-binding to endothelial Unc5B also controlled BRB integrity, and if this process involved Norrin/ß-catenin signaling, which is the major known driver of BRB development and maintenance. Methods: We analyzed Tamoxifen-inducible loss- and gain- of-function alleles of Unc5B, Ntn1 and Ctnnb1 in conjunction with tracer injections and biochemical signaling studies. Results: Inducible endothelial Unc5B deletion, and inducible global Ntn1 deletion in postnatal mice reduced phosphorylation of the Norrin receptor LRP5, leading to reduced ß-catenin and LEF1 expression, conversion of retina endothelial cells from a barrier-competent Claudin-5+/PLVAP- state to a Claudin-5-/PLVAP+ leaky phenotype, and extravasation of injected low molecular weight tracers. Inducible Ctnnb1 gain of function rescued vascular leak in Unc5B mutants, and Ntn1 overexpression induced BRB tightening. Unc5B expression in pericytes contributed to BRB permeability, via regulation of endothelial Unc5B. Mechanistically, Netrin-1-Unc5B signaling promoted ß-catenin dependent BRB signaling by enhancing phosphorylation of the Norrin receptor LRP5 via the Discs large homologue 1 (Dlg1) intracellular scaffolding protein. Conclusions: The data identify Netrin1-Unc5B as novel regulators of BRB integrity, with implications for diseases associated with BRB disruption.
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During blood vessel development, endothelial cells become specified toward arterial or venous fates to generate a circulatory network that provides nutrients and oxygen to, and removes metabolic waste from, all tissues. Arterial-venous specification occurs in conjunction with suppression of endothelial cell cycle progression; however, the mechanistic role of cell cycle state is unknown. Herein, using Cdh5-CreERT2;R26FUCCI2aR reporter mice, we find that venous endothelial cells are enriched for the FUCCI-Negative state (early G1) and BMP signaling, while arterial endothelial cells are enriched for the FUCCI-Red state (late G1) and TGF-ß signaling. Furthermore, early G1 state is essential for BMP4-induced venous gene expression, whereas late G1 state is essential for TGF-ß1-induced arterial gene expression. Pharmacologically induced cell cycle arrest prevents arterial-venous specification defects in mice with endothelial hyperproliferation. Collectively, our results show that distinct endothelial cell cycle states provide distinct windows of opportunity for the molecular induction of arterial vs. venous fate.
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Células Endoteliales , Factor de Crecimiento Transformador beta1 , Animales , Arterias/metabolismo , Ciclo Celular , Células Endoteliales/metabolismo , Ratones , Oxígeno/metabolismo , Factor de Crecimiento Transformador beta1/metabolismo , VenasRESUMEN
Blood-brain barrier (BBB) integrity is critical for proper function of the central nervous system (CNS). Here, we show that the endothelial Unc5B receptor controls BBB integrity by maintaining Wnt/ß-catenin signaling. Inducible endothelial-specific deletion of Unc5B in adult mice leads to BBB leak from brain capillaries that convert to a barrier-incompetent state with reduced Claudin-5 and increased PLVAP expression. Loss of Unc5B decreases BBB Wnt/ß-catenin signaling, and ß-catenin overexpression rescues Unc5B mutant BBB defects. Mechanistically, the Unc5B ligand Netrin-1 enhances Unc5B interaction with the Wnt co-receptor LRP6, induces its phosphorylation and activates Wnt/ß-catenin downstream signaling. Intravenous delivery of antibodies blocking Netrin-1 binding to Unc5B causes a transient BBB breakdown and disruption of Wnt signaling, followed by neurovascular barrier resealing. These data identify Netrin-1-Unc5B signaling as a ligand-receptor pathway that regulates BBB integrity, with implications for CNS diseases.
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Barrera Hematoencefálica , Receptores de Netrina , Animales , Barrera Hematoencefálica/metabolismo , Células Endoteliales/metabolismo , Ligandos , Ratones , Receptores de Netrina/genética , Receptores de Netrina/metabolismo , Netrina-1/genética , Netrina-1/metabolismo , Vía de Señalización Wnt , beta Catenina/metabolismoRESUMEN
BACKGROUND: Activin receptor-like kinase 1 (ALK1) is an endothelial transmembrane serine threonine kinase receptor for BMP family ligands that plays a critical role in cardiovascular development and pathology. Loss-of-function mutations in the ALK1 gene cause type 2 hereditary hemorrhagic telangiectasia, a devastating disorder that leads to arteriovenous malformations. Here, we show that ALK1 controls endothelial cell polarization against the direction of blood flow and flow-induced endothelial migration from veins through capillaries into arterioles. METHODS: Using Cre lines that recombine in different subsets of arterial, capillary-venous, or endothelial tip cells, we show that capillary-venous Alk1 deletion was sufficient to induce arteriovenous malformation formation in the postnatal retina. RESULTS: ALK1 deletion impaired capillary-venous endothelial cell polarization against the direction of blood flow in vivo and in vitro. Mechanistically, ALK1-deficient cells exhibited increased integrin signaling interaction with vascular endothelial growth factor receptor 2, which enhanced downstream YAP/TAZ nuclear translocation. Pharmacologic inhibition of integrin or YAP/TAZ signaling rescued flow migration coupling and prevented vascular malformations in Alk1-deficient mice. CONCLUSIONS: Our study reveals ALK1 as an essential driver of flow-induced endothelial cell migration and identifies loss of flow-migration coupling as a driver of arteriovenous malformation formation in hereditary hemorrhagic telangiectasia disease. Integrin-YAP/TAZ signaling blockers are new potential targets to prevent vascular malformations in patients with hereditary hemorrhagic telangiectasia.
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Malformaciones Arteriovenosas , Células Endoteliales , Telangiectasia Hemorrágica Hereditaria , Factor A de Crecimiento Endotelial Vascular , Animales , Humanos , Malformaciones Arteriovenosas/metabolismo , Movimiento Celular/fisiología , Células Endoteliales/metabolismo , Telangiectasia Hemorrágica Hereditaria/mortalidad , Factor A de Crecimiento Endotelial Vascular/metabolismo , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismo , Malformaciones Vasculares/metabolismo , RatonesRESUMEN
Protein tyrosine phosphatases are essential modulators of angiogenesis and have been identified as novel therapeutic targets in cancer and anti-angiogenesis. The roles of atypical Phosphatase of Regenerative Liver (PRL) phosphatases in this context remain poorly understood. Here, we investigate the biological function of PRL phosphatases in developmental angiogenesis in the postnatal mouse retina and in cell culture. We show that endothelial cells in the retina express PRL-2 encoded by the Ptp4a2 gene, and that inducible endothelial and global Ptp4a2 mutant mice exhibit defective retinal vascular outgrowth, arteriovenous differentiation, and sprouting angiogenesis. Mechanistically, PTP4A2 deletion limits angiogenesis by inhibiting endothelial cell migration and the VEGF-A, DLL-4/NOTCH-1 signaling pathway. This study reveals the importance of PRL-2 as a modulator of vascular development.