Advances and controversies in meningeal biology.

The dura, arachnoid and pia mater, as the constituent layers of the meninges, along with cerebrospinal fluid in the subarachnoid space and ventricles, are essential protectors of the brain and spinal cord. Complemented by immune cells, blood vessels, lymphatic vessels and nerves, these connective tissue layers have held many secrets that have only recently begun to be revealed. Each meningeal layer is now known to have molecularly distinct types of fibroblasts. Cerebrospinal fluid clearance through peripheral lymphatics and lymph nodes is well documented, but its routes and flow dynamics are debated. Advances made in meningeal immune functions are also debated. This Review considers the cellular and molecular structure and function of the dura, arachnoid and pia mater in the context of conventional views, recent progress, and what is uncertain or unknown. The hallmarks of meningeal pathophysiology are identified toward developing a more complete understanding of the meninges in health and disease.

A systems view of the vascular endothelium in health and disease.

The dysfunction of blood-vessel-lining endothelial cells is a major cause of mortality. Although endothelial cells, being present in all organs as a single-cell layer, are often conceived as a rather inert cell population, the vascular endothelium as a whole should be considered a highly dynamic and interactive systemically disseminated organ. We present here a holistic view of the field of vascular research and review the diverse functions of blood-vessel-lining endothelial cells during the life cycle of the vasculature, namely responsive and relaying functions of the vascular endothelium and the responsive roles as instructive gatekeepers of organ function. Emerging translational perspectives in regenerative medicine, preventive medicine, and aging research are developed. Collectively, this review is aimed at promoting disciplinary coherence in the field of angioscience for a broader appreciation of the importance of the vasculature for organ function, systemic health, and healthy aging.

Loss of Katnal2 leads to ependymal ciliary hyperfunction and autism-related phenotypes in mice.

Autism spectrum disorders (ASD) frequently accompany macrocephaly, which often involves hydrocephalic enlargement of brain ventricles. Katnal2 is a microtubule-regulatory protein strongly linked to ASD, but it remains unclear whether Katnal2 knockout (KO) in mice leads to microtubule- and ASD-related molecular, synaptic, brain, and behavioral phenotypes. We found that Katnal2-KO mice display ASD-like social communication deficits and age-dependent progressive ventricular enlargements. The latter involves increased length and beating frequency of motile cilia on ependymal cells lining ventricles. Katnal2-KO hippocampal neurons surrounded by enlarged lateral ventricles show progressive synaptic deficits that correlate with ASD-like transcriptomic changes involving synaptic gene down-regulation. Importantly, early postnatal Katnal2 re-expression prevents ciliary, ventricular, and behavioral phenotypes in Katnal2-KO adults, suggesting a causal relationship and a potential treatment. Therefore, Katnal2 negatively regulates ependymal ciliary function and its deletion in mice leads to ependymal ciliary hyperfunction and hydrocephalus accompanying ASD-related behavioral, synaptic, and transcriptomic changes.

Meningeal lymphatics can influence stroke outcome.

Meningeal lymphatics are conduits for cerebrospinal fluid drainage to lymphatics and lymph nodes in the neck. In this issue of JEM, Boisserand et al. (https://doi.org/10.1084/jem.20221983) provide evidence that expansion of meningeal lymphatics protects against ischemic stroke.

Fibroblasts enable penile erection.

Perivascular fibroblasts may underlie erectile dysfunction.

Nasopharyngeal lymphatic plexus is a hub for cerebrospinal fluid drainage.

Cerebrospinal fluid (CSF) in the subarachnoid space around the brain has long been known to drain through the lymphatics to cervical lymph nodes1-17, but the connections and regulation have been challenging to identify. Here, using fluorescent CSF tracers in Prox1-GFP lymphatic reporter mice18, we found that the nasopharyngeal lymphatic plexus is a major hub for CSF outflow to deep cervical lymph nodes. This plexus had unusual valves and short lymphangions but no smooth-muscle coverage, whereas downstream deep cervical lymphatics had typical semilunar valves, long lymphangions and smooth muscle coverage that transported CSF to the deep cervical lymph nodes. α-Adrenergic and nitric oxide signalling in the smooth muscle cells regulated CSF drainage through the transport properties of deep cervical lymphatics. During ageing, the nasopharyngeal lymphatic plexus atrophied, but deep cervical lymphatics were not similarly altered, and CSF outflow could still be increased by adrenergic or nitric oxide signalling. Single-cell analysis of gene expression in lymphatic endothelial cells of the nasopharyngeal plexus of aged mice revealed increased type I interferon signalling and other inflammatory cytokines. The importance of evidence for the nasopharyngeal lymphatic plexus functioning as a CSF outflow hub is highlighted by its regression during ageing. Yet, the ageing-resistant pharmacological activation of deep cervical lymphatic transport towards lymph nodes can still increase CSF outflow, offering an approach for augmenting CSF clearance in age-related neurological conditions in which greater efflux would be beneficial.

Immaturity of immune cells around the dural venous sinuses contributes to viral meningoencephalitis in neonates.

High neonatal susceptibility to meningitis has been attributed to the anatomical barriers that act to protect the central nervous system (CNS) from infection being immature and not fully developed. However, the mechanisms by which pathogens breach CNS barriers are poorly understood. Using the Armstrong strain of lymphocytic choriomeningitis virus (LCMV) to study virus propagation into the CNS during systemic infection, we demonstrate that mortality in neonatal, but not adult, mice is high after infection. Virus propagated extensively from the perivenous sinus region of the dura mater to the leptomeninges, choroid plexus, and cerebral cortex. Although the structural barrier of CNS border tissues is comparable between neonates and adults, immunofluorescence staining and single-cell RNA sequencing analyses revealed that the neonatal dural immune cells are immature and predominantly composed of CD206hi macrophages, with major histocompatibility complex class II (MHCII)hi macrophages being rare. In adults, however, perivenous sinus immune cells were enriched in MHCIIhi macrophages that are specialized for producing antiviral molecules and chemokines compared with CD206hi macrophages and protected the CNS against systemic virus invasion. Our findings clarify how systemic pathogens enter the CNS through its border tissues and how the immune barrier at the perivenous sinus region of the dura blocks pathogen access to the CNS.

Endothelial cell-derived stem cell factor promotes lipid accumulation through c-Kit-mediated increase of lipogenic enzymes in brown adipocytes.

Active thermogenesis in the brown adipose tissue (BAT) facilitating the utilization of lipids and glucose is critical for maintaining body temperature and reducing metabolic diseases, whereas inactive BAT accumulates lipids in brown adipocytes (BAs), leading to BAT whitening. Although cellular crosstalk between endothelial cells (ECs) and adipocytes is essential for the transport and utilization of fatty acid in BAs, the angiocrine roles of ECs mediating this crosstalk remain poorly understood. Using single-nucleus RNA sequencing and knock-out male mice, we demonstrate that stem cell factor (SCF) derived from ECs upregulates gene expressions and protein levels of the enzymes for de novo lipogenesis, and promotes lipid accumulation by activating c-Kit in BAs. In the early phase of lipid accumulation induced by denervation or thermoneutrality, transiently expressed c-Kit on BAs increases the protein levels of the lipogenic enzymes via PI3K and AKT signaling. EC-specific SCF deletion and BA-specific c-Kit deletion attenuate the induction of the lipogenic enzymes and suppress the enlargement of lipid droplets in BAs after denervation or thermoneutrality in male mice. These data provide insight into SCF/c-Kit signaling as a regulator that promotes lipid accumulation through the increase of lipogenic enzymes in BAT when thermogenesis is inhibited.

Hyperactivation of YAP/TAZ Drives Alterations in Mesangial Cells through Stabilization of N-Myc in Diabetic Nephropathy.

SIGNIFICANCE STATEMENT: Mesangial cells (MCs) in the kidney are essential to maintaining glomerular integrity, and their impairment leads to major glomerular diseases including diabetic nephropathy (DN). Although high blood glucose elicits abnormal alterations in MCs, the underlying mechanism is poorly understood. We show that YAP/TAZ are increased in MCs of patients with DN and two animal models of DN. High glucose directly induces activation of YAP/TAZ through the canonical Hippo pathway in cultured MCs. Hyperactivation of YAP/TAZ in mouse MCs recapitulates the hallmarks of DN. Activated YAP/TAZ bind and stabilize N-Myc, one of the Myc family. N-Myc stabilization leads to aberrant enhancement of its transcriptional activity and to MC impairments. Our findings shed light on how high blood glucose in diabetes mellitus leads to DN and support a rationale that lowering blood glucose in diabetes mellitus could delay DN pathogenesis. BACKGROUND: Mesangial cells (MCs) in the kidney are central to maintaining glomerular integrity, and their impairment leads to major glomerular diseases, including diabetic nephropathy (DN). Although high blood glucose elicits abnormal alterations in MCs, the underlying molecular mechanism is poorly understood. METHODS: Immunolocalization of YAP/TAZ and pathological features of PDGFRß + MCs were analyzed in the glomeruli of patients with DN, in Zucker diabetic fatty rats, and in Lats1/2i ΔPß mice. RiboTag bulk-RNA sequencing and transcriptomic analysis of gene expression profiles of the isolated MCs from control and Lats1/2iΔPß mice were performed. Immunoprecipitation analysis and protein stability of N-Myc were performed by the standard protocols. RESULTS: YAP and TAZ, the final effectors of the Hippo pathway, are highly increased in MCs of patients with DN and in Zucker diabetic fatty rats. Moreover, high glucose directly induces activation of YAP/TAZ through the canonical Hippo pathway in cultured MCs. Hyperactivation of YAP/TAZ in mouse model MCs recapitulates the hallmarks of DN, including excessive proliferation of MCs and extracellular matrix deposition, endothelial cell impairment, glomerular sclerosis, albuminuria, and reduced glomerular filtration rate. Mechanistically, activated YAP/TAZ bind and stabilize N-Myc protein, one of the Myc family of oncogenes. N-Myc stabilization leads to aberrant enhancement of its transcriptional activity and eventually to MC impairments and DN pathogenesis. CONCLUSIONS: Our findings shed light on how high blood glucose in diabetes mellitus leads to DN and support a rationale that lowering blood glucose in diabetes mellitus could delay DN pathogenesis.

Fasudil alleviates the vascular endothelial dysfunction and several phenotypes of Fabry disease.

Fabry disease (FD), a lysosomal storage disorder, is caused by defective α-galactosidase (GLA) activity, which results in the accumulation of globotriaosylceramide (Gb3) in endothelial cells and leads to life-threatening complications such as left ventricular hypertrophy (LVH), renal failure, and stroke. Enzyme replacement therapy (ERT) results in Gb3 clearance; however, because of a short half-life in the body and the high immunogenicity of FD patients, ERT has a limited therapeutic effect, particularly in patients with late-onset disease or progressive complications. Because vascular endothelial cells (VECs) derived from FD-induced pluripotent stem cells display increased thrombospondin-1 (TSP1) expression and enhanced SMAD2 signaling, we screened for chemical compounds that could downregulate TSP1 and SMAD2 signaling. Fasudil reduced the levels of p-SMAD2 and TSP1 in FD-VECs and increased the expression of angiogenic factors. Furthermore, fasudil downregulated the endothelial-to-mesenchymal transition (EndMT) and mitochondrial function of FD-VECs. Oral administration of fasudil to FD mice alleviated several FD phenotypes, including LVH, renal fibrosis, anhidrosis, and heat insensitivity. Our findings demonstrate that fasudil is a novel candidate for FD therapy.

Three-dimensional morphologic and molecular atlases of nasal vasculature.

Understanding the function of the nasal vasculature in homeostasis and pathogenesis of common nasal diseases is important. Here we describe an extensive network of venous sinusoids (VSs) in mouse and human nasal mucosa. The endothelium of the VSs expressed Prox1 (considered to be a constitutive marker of lymphatic endothelium) and high levels of VCAM-1 and exhibited unusual cell-to-cell junctions. VSs are supported by circular smooth muscle cells (SMCs) and surrounded by immune cells. The nasal mucosa also showed a rich supply of lymphatic vessels with distinctive features, such as the absence of the lymphatic marker LYVE1 and sharp-ended capillaries. In mouse models of allergic rhinitis or acute Coronavirus Disease 2019 (COVID-19) infection, Prox1+ VSs were regressed or compromised. However, in aged mice, the VSs lost the SMC support and were expanded and enlarged. Our findings demonstrate three-dimensional morphological and molecular heterogeneities of the nasal vasculature and offer insights into their associations with nasal inflammation, infection and aging.

Gatekeeping role of Nf2/Merlin in vascular tip EC induction through suppression of VEGFR2 internalization.

In sprouting angiogenesis, the precise mechanisms underlying how intracellular vascular endothelial growth factor receptor 2 (VEGFR2) signaling is higher in one endothelial cell (EC) compared with its neighbor and acquires the tip EC phenotype under a similar external cue are elusive. Here, we show that Merlin, encoded by the neurofibromatosis type 2 (NF2) gene, suppresses VEGFR2 internalization depending on VE-cadherin density and inhibits tip EC induction. Accordingly, endothelial Nf2 depletion promotes tip EC induction with excessive filopodia by enhancing VEGFR2 internalization in both the growing and matured vessels. Mechanistically, Merlin binds to the VEGFR2-VE-cadherin complex at cell-cell junctions and reduces VEGFR2 internalization-induced downstream signaling during tip EC induction. As a consequence, nonfunctional excessive sprouting occurs during tumor angiogenesis in EC-specific Nf2-deleted mice, leading to delayed tumor growth. Together, Nf2/Merlin is a crucial molecular gatekeeper for tip EC induction, capillary integrity, and proper tumor angiogenesis by suppressing VEGFR2 internalization.

Pericyte Loss Leads to Capillary Stalling Through Increased Leukocyte-Endothelial Cell Interaction in the Brain.

The neurovascular unit is a functional unit composed of neurons, glial cells, pericytes, and endothelial cells which sustain brain activity. While pericyte is a key component of the neurovascular unit, its role in cerebral blood flow regulation remains elusive. Recently, capillary stalling, which means the transient interruption of microcirculation in capillaries, has been shown to have an outsized impact on microcirculatory changes in several neurological diseases. In this study, we investigated capillary stalling and its possible causes, such as the cerebral endothelial glycocalyx and leukocyte adhesion molecules after depleting pericytes postnatally in mice. Moreover, we investigated hypoxia and gliosis as consequences of capillary stalling. Although there were no differences in the capillary structure and RBC flow, longitudinal optical coherence tomography angiography showed an increased number of stalled segments in capillaries after pericyte loss. Furthermore, the extent of the cerebral endothelial glycocalyx was decreased with increased expression of leukocyte adhesion molecules, suggesting enhanced interaction between leukocytes and endothelial cells. Finally, pericyte loss induced cerebral hypoxia and gliosis. Cumulatively, the results suggest that pericyte loss induces capillary stalling through increased interaction between leukocytes and endothelial cells in the brain.

Viewpoints: Dual-blocking antibody against VEGF-A and angiopoietin-2 for treating vascular diseases of the eye.

Faricimab, a bispecific antibody that targets the endothelial cell growth factors vascular endothelial growth factor-A (VEGF-A) and angiopoietin-2 (Angpt2), was recently approved for treating neovascular age-related macular degeneration and diabetic macular edema. Here, Koh and Augustin review how mechanistic studies have translated into therapies, while Campochiaro evaluates their impact and value for clinical practice.

Antiangiogenesis: Vessel Regression, Vessel Normalization, or Both?

The concepts of antiangiogenic tumor therapy were pioneered on the assumption that the inhibition of tumor angiogenesis should lead to the complete regression of the tumor-associated vasculature and thereby hold the tumor in an avascular dormant state. Yet, clinical trials revealed limited efficacy of angiogenesis inhibitors when used as monotherapy. Instead, antiangiogenic drugs proved effective to extend overall survival when used in combination with chemotherapy. This counterintuitive observation-inhibition of tumor vascularization should lead to less and not more delivery of chemotherapy to the tumor-led to the concepts of "vessel normalization." This refers to the notion that antiangiogenic drugs prune the most immature tumor vessels and spare mature vessels, thereby resulting in a more normal-appearing vasculature that leads to better access of chemotherapy to the tumor. The concepts of vessel normalization were first laid out in a landmark publication in Cancer Research in 2004. More than 600 studies on different aspects of vessel normalization have been published since then. Nevertheless, it is to this day less clear than ever to what extent vessel regression (leading to tumor starvation) and vessel normalization (facilitating chemotherapy) contribute to the clinical efficacy of antiangiogenic tumor therapy. This "Landmark Commentary" puts the concepts of tumor vessel normalization in historical context and develops thereupon some of the most burning questions in the field of translational angiogenesis research that need to be answered to further advance the application of tumor vascular stroma reprogramming therapies.See related article by Tong and colleagues, Cancer Res 2004;64:3731-6.

CHD4 Conceals Aberrant CTCF-Binding Sites at TAD Interiors by Regulating Chromatin Accessibility in Mouse Embryonic Stem Cells.

CCCTC-binding factor (CTCF) critically contributes to 3D chromatin organization by determining topologically associated domain (TAD) borders. Although CTCF primarily binds at TAD borders, there also exist putative CTCF-binding sites within TADs, which are spread throughout the genome by retrotransposition. However, the detailed mechanism responsible for masking the putative CTCF-binding sites remains largely elusive. Here, we show that the ATP-dependent chromatin remodeler, chromodomain helicase DNA-binding 4 (CHD4), regulates chromatin accessibility to conceal aberrant CTCF-binding sites embedded in H3K9me3-enriched heterochromatic B2 short interspersed nuclear elements (SINEs) in mouse embryonic stem cells (mESCs). Upon CHD4 depletion, these aberrant CTCF-binding sites become accessible and aberrant CTCF recruitment occurs within TADs, resulting in disorganization of local TADs. RNA-binding intrinsically disordered domains (IDRs) of CHD4 are required to prevent this aberrant CTCF binding, and CHD4 is critical for the repression of B2 SINE transcripts. These results collectively reveal that a CHD4-mediated mechanism ensures appropriate CTCF binding and associated TAD organization in mESCs.

Structural insights into the clustering and activation of Tie2 receptor mediated by Tie2 agonistic antibody.

Angiopoietin (Angpt)-Tie receptor 2 (Tie2) plays key roles in vascular development and homeostasis as well as pathological vascular remodeling. Therefore, Tie2-agonistic antibody and engineered Angpt1 variants have been developed as potential therapeutics for ischemic and inflammatory vascular diseases. However, their underlying mechanisms for Tie2 clustering and activation remain elusive and the poor manufacturability and stability of Angpt1 variants limit their clinical application. Here, we develop a human Tie2-agonistic antibody (hTAAB), which targets the membrane proximal fibronectin type III domain of Tie2 distinct from the Angpt-binding site. Our Tie2/hTAAB complex structures reveal that hTAAB tethers the preformed Tie2 homodimers into polygonal assemblies through specific binding to Tie2 Fn3 domain. Notably, the polygonal Tie2 clustering induced by hTAAB is critical for Tie2 activation and are resistant to antagonism by Angpt2. Our results provide insight into the molecular mechanism of Tie2 clustering and activation mediated by hTAAB, and the structure-based humanization of hTAAB creates a potential clinical application.