Meningeal lymphatic function promotes oligodendrocyte survival and brain myelination.

The precise neurophysiological changes prompted by meningeal lymphatic dysfunction remain unclear. Here, we showed that inducing meningeal lymphatic vessel ablation in adult mice led to gene expression changes in glial cells, followed by reductions in mature oligodendrocyte numbers and specific lipid species in the brain. These phenomena were accompanied by altered meningeal adaptive immunity and brain myeloid cell activation. During brain remyelination, meningeal lymphatic dysfunction provoked a state of immunosuppression in the brain that contributed to delayed spontaneous oligodendrocyte replenishment and axonal loss. The deficiencies in mature oligodendrocytes and neuroinflammation due to impaired meningeal lymphatic function were solely recapitulated in immunocompetent mice. Patients diagnosed with multiple sclerosis presented reduced vascular endothelial growth factor C in the cerebrospinal fluid, particularly shortly after clinical relapses, possibly indicative of poor meningeal lymphatic function. These data demonstrate that meningeal lymphatics regulate oligodendrocyte function and brain myelination, which might have implications for human demyelinating diseases.

Meningeal lymphatic vessel dysfunction driven by CGRP signaling causes migraine-like pain in mice.

Migraines are a type of headache that occur with other neurological symptoms, but the pathophysiology remains unclear. In this issue of the JCI, Nelson-Maney and authors used constitutive and inducible knockouts of the CGRP receptor components, elegantly demonstrating an essential function of CGRP in modulating meningeal lymphatic vessels (MLVs) in migraine. CGRP was shown to induce rearrangement of membrane-bound gap junction proteins in MLVs, resulting in a reduced CSF flux into cervical lymph nodes. The authors also provided evidence of a primary role for CGRP in modulating neuro-immune function. Finally, by showing that blocking CGRP signaling in MLVs attenuated pain behavior associated with acute migraine in rodents, the authors provided a target for pharmacological blockade of CGRP in relation to primary headache disorders.

Complementary and Inducible creERT2 Mouse Models for Functional Evaluation of Endothelial Cell Subtypes in the Bone Marrow.

In the adult bone marrow (BM), endothelial cells (ECs) are an integral component of the hematopoietic stem cell (HSC)-supportive niche, which modulates HSC activity by producing secreted and membrane-bound paracrine signals. Within the BM, distinct vascular arteriole, transitional, and sinusoidal EC subtypes display unique paracrine expression profiles and create anatomically-discrete microenvironments. However, the relative contributions of vascular endothelial subtypes in supporting hematopoiesis is unclear. Moreover, constitutive expression and off-target activity of currently available endothelial-specific and endothelial-subtype-specific murine cre lines potentially confound data analysis and interpretation. To address this, we describe two tamoxifen-inducible cre-expressing lines, Vegfr3-creERT2 and Cx40-creERT2, that efficiently label sinusoidal/transitional and arteriole endothelium respectively in adult marrow, without off-target activity in hematopoietic or perivascular cells. Utilizing an established mouse model in which cre-dependent recombination constitutively-activates MAPK signaling within adult endothelium, we identify arteriole ECs as the driver of MAPK-mediated hematopoietic dysfunction. These results define complementary tamoxifen-inducible creERT2-expressing mouse lines that label functionally-discrete and non-overlapping sinusoidal/transitional and arteriole EC populations in the adult BM, providing a robust toolset to investigate the differential contributions of vascular subtypes in maintaining hematopoietic homeostasis.

VEGF-C prophylaxis favors lymphatic drainage and modulates neuroinflammation in a stroke model.

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.

Compartmentalized ocular lymphatic system mediates eye-brain immunity.

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.

Intrathecal delivery of nanoparticle PARP inhibitor to the cerebrospinal fluid for the treatment of metastatic medulloblastoma.

The morbidity associated with pediatric medulloblastoma, in particular in patients who develop leptomeningeal metastases, remains high in the absence of effective therapies. Administration of substances directly into the cerebrospinal fluid (CSF) is one approach to circumvent the blood-brain barrier and focus delivery of drugs to the site of tumor. However, high rates of CSF turnover prevent adequate drug accumulation and lead to rapid systemic clearance and toxicity. Here, we show that PLA-HPG nanoparticles, made with a single-emulsion, solvent evaporation process, can encapsulate talazoparib, a PARP inhibitor (BMN-673). These degradable polymer nanoparticles improve the therapeutic index when delivered intrathecally and lead to sustained drug retention in the tumor as measured with PET imaging and fluorescence microscopy. We demonstrate that administration of these particles into the CSF, alone or in combination with systemically administered temozolomide, is a highly effective therapy for tumor regression and prevention of leptomeningeal spread in xenograft mouse models of medulloblastoma. These results provide a rationale for harnessing nanoparticles for the delivery of drugs limited by brain penetration and therapeutic index and demonstrate important advantages in tolerability and efficacy for encapsulated drugs delivered locoregionally.

VEGF-C promotes brain-derived fluid drainage, confers neuroprotection, and improves stroke outcomes.

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.

CCL21-CCR7 signaling promotes microglia/macrophage recruitment and chemotherapy resistance in glioblastoma.

Glioblastoma (GBM) is the most common and fatal primary tumor of the central nervous system (CNS) and current treatments have limited success. Chemokine signaling regulates both malignant cells and stromal cells of the tumor microenvironment (TME), constituting a potential therapeutic target against brain cancers. Here, we investigated the C-C chemokine receptor type 7 (CCR7) and the chemokine (C-C-motif) ligand 21 (CCL21) for their expression and function in human GBM and then assessed their therapeutic potential in preclinical mouse GBM models. In GBM patients, CCR7 expression positively associated with a poor survival. CCL21-CCR7 signaling was shown to regulate tumor cell migration and proliferation while also controlling tumor associated microglia/macrophage recruitment and VEGF-A production, thereby controlling vascular dysmorphia. Inhibition of CCL21-CCR7 signaling led to an increased sensitivity to temozolomide-induced tumor cell death. Collectively, our data indicate that drug targeting of CCL21-CCR7 signaling in tumor and TME cells is a therapeutic option against GBM.

Three-dimensional imaging of vascular development in the mouse epididymis.

Long considered an accessory tubule of the male reproductive system, the epididymis is proving to be a key determinant of male fertility. In addition to its secretory role in ensuring functional maturation and survival of spermatozoa, the epididymis has a complex immune function. Indeed, it must manage both peripheral tolerance to sperm antigens foreign to the immune system and the protection of spermatozoa as well as the organ itself against pathogens ascending the epididymal tubule. Although our knowledge of the immunobiology of this organ is beginning to accumulate at the molecular and cellular levels, the organization of blood and lymphatic networks of this tissue, important players in the immune response, remains largely unknown. In the present report, we have taken advantage of a VEGFR3:YFP transgenic mouse model. Using high-resolution three-dimensional (3D) imaging and organ clearing coupled with multiplex immunodetections of lymphatic (LYVE1, PDPN, PROX1) and/or blood (PLVAP/Meca32) markers, we provide a simultaneous deep 3D view of the lymphatic and blood epididymal vasculature in the mature adult mouse as well as during postnatal development.

Connexin 43-mediated neurovascular interactions regulate neurogenesis in the adult brain subventricular zone.

The subventricular zone (SVZ) is the largest neural stem cell (NSC) niche in the adult brain; herein, the blood-brain barrier is leaky, allowing direct interactions between NSCs and endothelial cells (ECs). Mechanisms by which direct NSC-EC interactions in the adult SVZ control NSC behavior are unclear. We found that Cx43 is highly expressed by SVZ NSCs and ECs, and its deletion in either leads to increased NSC proliferation and neuroblast generation, suggesting that Cx43-mediated NSC-EC interactions maintain NSC quiescence. This is further supported by single-cell RNA sequencing and in vitro studies showing that ECs control NSC proliferation by regulating expression of genes associated with NSC quiescence and/or activation in a Cx43-dependent manner. Cx43 mediates these effects in a channel-independent manner involving its cytoplasmic tail and ERK activation. Such insights inform adult NSC regulation and maintenance aimed at stem cell therapies for neurodegenerative disorders.

CSF-to-dura gateways.

Postmortem microstructural studies together with in vivo magnetic resonance imaging show that human arachnoid granulations are porous channels that serve as transient filtration conduits for cerebrospinal fluid to flow directly into dural interstitial tissue, but not into venous sinuses (Shah et al. 2022. J. Exp. Med.https://doi.org/10.1084/jem.20220618).

Conserved meningeal lymphatic drainage circuits in mice and humans.

Meningeal lymphatic vessels (MLVs) were identified in the dorsal and caudobasal regions of the dura mater, where they ensure waste product elimination and immune surveillance of brain tissues. Whether MLVs exist in the anterior part of the murine and human skull and how they connect with the glymphatic system and extracranial lymphatics remained unclear. Here, we used light-sheet fluorescence microscopy (LSFM) imaging of mouse whole-head preparations after OVA-A555 tracer injection into the cerebrospinal fluid (CSF) and performed real-time vessel-wall (VW) magnetic resonance imaging (VW-MRI) after systemic injection of gadobutrol in patients with neurological pathologies. We observed a conserved three-dimensional anatomy of MLVs in mice and humans that aligned with dural venous sinuses but not with nasal CSF outflow, and we discovered an extended anterior MLV network around the cavernous sinus, with exit routes through the foramina of emissary veins. VW-MRI may provide a diagnostic tool for patients with CSF drainage defects and neurological diseases.

Identification of growth hormone receptor as a relevant target for precision medicine in low-EGFR expressing glioblastoma.

OBJECTIVE: New therapeutic approaches are needed to improve the prognosis of glioblastoma (GBM) patients. METHODS: With the objective of identifying alternative oncogenic mechanisms to abnormally activated epidermal growth factor receptor (EGFR) signalling, one of the most common oncogenic mechanisms in GBM, we performed a comparative analysis of gene expression profiles in a series of 54 human GBM samples. We then conducted gain of function as well as genetic and pharmocological inhibition assays in GBM patient-derived cell lines to functionnally validate our finding. RESULTS: We identified that growth hormone receptor (GHR) signalling defines a distinct molecular subset of GBMs devoid of EGFR overexpression. GHR overexpression was detected in one third of patients and was associated with low levels of suppressor of cytokine signalling 2 (SOCS2) expression due to SOCS2 promoter hypermethylation. In GBM patient-derived cell lines, GHR signalling modulates the expression of proteins involved in cellular movement, promotes cell migration, invasion and proliferation in vitro and promotes tumourigenesis, tumour growth, and tumour invasion in vivo. GHR genetic and pharmacological inhibition reduced cell proliferation and migration in vitro. CONCLUSION: This study pioneers a new field of investigation to improve the prognosis of GBM patients.

SLIT2/ROBO signaling in tumor-associated microglia and macrophages drives glioblastoma immunosuppression and vascular dysmorphia.

SLIT2 is a secreted polypeptide that guides migration of cells expressing Roundabout 1 and 2 (ROBO1 and ROBO2) receptors. Herein, we investigated SLIT2/ROBO signaling effects in gliomas. In patients with glioblastoma (GBM), SLIT2 expression increased with malignant progression and correlated with poor survival and immunosuppression. Knockdown of SLIT2 in mouse glioma cells and patient-derived GBM xenografts reduced tumor growth and rendered tumors sensitive to immunotherapy. Tumor cell SLIT2 knockdown inhibited macrophage invasion and promoted a cytotoxic gene expression profile, which improved tumor vessel function and enhanced efficacy of chemotherapy and immunotherapy. Mechanistically, SLIT2 promoted microglia/macrophage chemotaxis and tumor-supportive polarization via ROBO1- and ROBO2-mediated PI3K-γ activation. Macrophage Robo1 and Robo2 deletion and systemic SLIT2 trap delivery mimicked SLIT2 knockdown effects on tumor growth and the tumor microenvironment (TME), revealing SLIT2 signaling through macrophage ROBOs as a potentially novel regulator of the GBM microenvironment and immunotherapeutic target for brain tumors.