Meningeal Immunity and Its Function in Maintenance of the Central Nervous System in Health and Disease.

Neuroimmunology, albeit a relatively established discipline, has recently sparked numerous exciting findings on microglia, the resident macrophages of the central nervous system (CNS). This review addresses meningeal immunity, a less-studied aspect of neuroimmune interactions. The meninges, a triple layer of membranes-the pia mater, arachnoid mater, and dura mater-surround the CNS, encompassing the cerebrospinal fluid produced by the choroid plexus epithelium. Unlike the adjacent brain parenchyma, the meninges contain a wide repertoire of immune cells. These constitute meningeal immunity, which is primarily concerned with immune surveillance of the CNS, and-according to recent evidence-also participates in postinjury CNS recovery, chronic neurodegenerative conditions, and even higher brain function. Meningeal immunity has recently come under the spotlight owing to the characterization of meningeal lymphatic vessels draining the CNS. Here, we review the current state of our understanding of meningeal immunity and its effects on healthy and diseased brains.

Lymphatic vessels in bone support regeneration after injury.

Blood and lymphatic vessels form a versatile transport network and provide inductive signals to regulate tissue-specific functions. Blood vessels in bone regulate osteogenesis and hematopoiesis, but current dogma suggests that bone lacks lymphatic vessels. Here, by combining high-resolution light-sheet imaging and cell-specific mouse genetics, we demonstrate presence of lymphatic vessels in mouse and human bones. We find that lymphatic vessels in bone expand during genotoxic stress. VEGF-C/VEGFR-3 signaling and genotoxic stress-induced IL6 drive lymphangiogenesis in bones. During lymphangiogenesis, secretion of CXCL12 from proliferating lymphatic endothelial cells is critical for hematopoietic and bone regeneration. Moreover, lymphangiocrine CXCL12 triggers expansion of mature Myh11+ CXCR4+ pericytes, which differentiate into bone cells and contribute to bone and hematopoietic regeneration. In aged animals, such expansion of lymphatic vessels and Myh11-positive cells in response to genotoxic stress is impaired. These data suggest lymphangiogenesis as a therapeutic avenue to stimulate hematopoietic and bone regeneration.

Lymphangiogenesis: Origin, Specification, and Cell Fate Determination.

The two vascular systems of our body are the blood and the lymphatic vasculature. Our understanding of the genes and molecular mechanisms controlling the development of the lymphatic vasculature network has significantly improved. The availability of novel animal models and better imaging tools led to the identification of lymphatics in tissues and organs previously thought to be devoid of them. Similarly, the classical textbook list of established functional roles of the lymphatic system has been expanded by the addition of novel findings. In this review we provide a historical perspective of some of the important landmarks that opened the doors to researchers working in this field. We also summarize some of the current views about embryonic lymphangiogenesis, particularly about the source(s), commitment, and differentiation of lymphatic endothelial cells.

Single-Cell Survey of Human Lymphatics Unveils Marked Endothelial Cell Heterogeneity and Mechanisms of Homing for Neutrophils.

Lymphatic vessels form a critical component in the regulation of human health and disease. While their functional significance is increasingly being recognized, the comprehensive heterogeneity of lymphatics remains uncharacterized. Here, we report the profiling of 33,000 lymphatic endothelial cells (LECs) in human lymph nodes (LNs) by single-cell RNA sequencing. Unbiased clustering revealed six major types of human LECs. LECs lining the subcapsular sinus (SCS) of LNs abundantly expressed neutrophil chemoattractants, whereas LECs lining the medullary sinus (MS) expressed a C-type lectin CD209. Binding of a carbohydrate Lewis X (CD15) to CD209 mediated neutrophil binding to the MS. The neutrophil-selective homing by MS LECs may retain neutrophils in the LN medulla and allow lymph-borne pathogens to clear, preventing their spread through LNs in humans. Our study provides a comprehensive characterization of LEC heterogeneity and unveils a previously undefined role for medullary LECs in human immunity.

Decorin suppresses tumor lymphangiogenesis: A mechanism to curtail cancer progression.

The complex interplay between malignant cells and the cellular and molecular components of the tumor stroma is a key aspect of cancer growth and development. These tumor-host interactions are often affected by soluble bioactive molecules such as proteoglycans. Decorin, an archetypical small leucine-rich proteoglycan primarily expressed by stromal cells, affects cancer growth in its soluble form by interacting with several receptor tyrosine kinases (RTK). Overall, decorin leads to a context-dependent and protracted cessation of oncogenic RTK activity by attenuating their ability to drive a prosurvival program and to sustain a proangiogenic network. Through an unbiased transcriptomic analysis using deep RNAseq, we identified that decorin down-regulated a cluster of tumor-associated genes involved in lymphatic vessel (LV) development when systemically delivered to mice harboring breast carcinoma allografts. We found that Lyve1 and Podoplanin, two established markers of LVs, were markedly suppressed at both the mRNA and protein levels, and this suppression correlated with a significant reduction in tumor LVs. We further identified that soluble decorin, but not its homologous proteoglycan biglycan, inhibited LV sprouting in an ex vivo 3D model of lymphangiogenesis. Mechanistically, we found that decorin interacted with vascular endothelial growth factor receptor 3 (VEGFR3), the main lymphatic RTK, and its activity was required for the decorin-mediated block of lymphangiogenesis. Finally, we identified that Lyve1 was in part degraded via decorin-evoked autophagy in a nutrient- and energy-independent manner. These findings implicate decorin as a biological factor with antilymphangiogenic activity and provide a potential therapeutic agent for curtailing breast cancer growth and metastasis.

Immune response after central nervous system injury.

Traumatic injuries of the central nervous system (CNS) affect millions of people worldwide, and they can lead to severely damaging consequences such as permanent disability and paralysis. Multiple factors can obstruct recovery after CNS injury. One of the most significant is the progressive neuronal death that follows the initial mechanical impact, leading to the loss of undamaged cells via a process termed secondary neurodegeneration. Efforts to define treatments that limit the spread of damage, while important, have been largely ineffectual owing to gaps in the mechanistic understanding that underlies the persisting neuronal cell death. Inflammation, with its influx of immune cells that occurs shortly after injury, has been associated with secondary neurodegeneration. However, the role of the immune system after CNS injury is far more complex. Studies have indicated that the immune response after CNS injury is detrimental, owing to immune cell-produced factors (e.g., pro-inflammatory cytokines, free radicals, neurotoxic glutamate) that worsen tissue damage. Our lab and others have also demonstrated the beneficial immune response that occurs after CNS injury, with the release of growth factors such as brain-derived growth factor (BDNF) and interleukin (IL-10) and the clearance of apoptotic and myelin debris by immune cells1-4. In this review, we first discuss the multifaceted roles of the immune system after CNS injury. We then speculate on how advancements in single-cell RNA technologies can dramatically change our understanding of the immune response, how the spinal cord meninges serve as an important site for hosting immunological processes critical for recovery, and how the origin of peripherally recruited immune cells impacts their function in the injured CNS.

Regulation of autoimmune-mediated neuroinflammation by endothelial cells.

The CNS has traditionally been considered an immune-privileged organ, but recent studies have identified a plethora of immune cells in the choroid plexus, meninges, perivascular spaces, and cribriform plate. Although those immune cells are crucial for the maintenance of CNS homeostasis and for neural protection against infections, they can lead to neuroinflammation in some circumstances. The blood and the lymphatic vasculatures exhibit distinct structural and molecular features depending on their location in the CNS, greatly influencing the compartmentalization and the nature of CNS immune responses. In this review, we discuss how endothelial cells regulate the migration and the functions of T cells in the CNS both at steady-state and in murine models of neuroinflammation, with a special focus on the anatomical, cellular, and molecular mechanisms implicated in EAE.

Brain vascular damage-induced lymphatic ingrowth is directed by Cxcl12b/Cxcr4a.

After ischemic stroke, promotion of vascular regeneration without causing uncontrolled vessel growth appears to be the major challenge for pro-angiogenic therapies. The molecular mechanisms underlying how nascent blood vessels (BVs) are correctly guided into the post-ischemic infarction area remain unknown. Here, using a zebrafish cerebrovascular injury model, we show that chemokine signaling provides crucial guidance cues to determine the growing direction of ingrown lymphatic vessels (iLVs) and, in turn, that of nascent BVs. The chemokine receptor Cxcr4a is transcriptionally activated in the iLVs after injury, whereas its ligand Cxcl12b is expressed in the residual central BVs, the destinations of iLV ingrowth. Mutant and mosaic studies indicate that Cxcl12b/Cxcr4a-mediated chemotaxis is necessary and sufficient to determine the growing direction of iLVs and nascent BVs. This study provides a molecular basis for how the vessel directionality of cerebrovascular regeneration is properly determined, suggesting potential application of Cxcl12b/Cxcr4a in the development of post-ischemic pro-angiogenic therapies.

The Lymphatic Vasculature in Cardiac Development and Ischemic Heart Disease.

In recent years, the lymphatic system has received increasing attention due to the fast-growing number of findings about its diverse novel functional roles in health and disease. It is well documented that the lymphatic vasculature plays major roles in the maintenance of tissue-fluid balance, the immune response, and in lipid absorption. However, recent studies have identified an additional growing number of novel and sometimes unexpected functional roles of the lymphatic vasculature in normal and pathological conditions in different organs. Among those, cardiac lymphatics have been shown to play important roles in heart development, ischemic cardiac disease, and cardiac disorders. In this review, we will discuss some of those novel functional roles of cardiac lymphatics, as well as the therapeutic potential of targeting lymphatics for the treatment of cardiovascular diseases.

Andrographolide promotes lymphangiogenesis and lymphatic vessel remodeling to alleviate secondary lymphedema.

Lymphedema, a prevalent, multifaceted, and chronic ailment, is mainly managed through physical manipulation and suffers from a lack of specific pharmacological treatments. Secondary lymphedema is mainly caused by impaired lymphatic drainage. Therapeutic lymphangiogenesis is a promising strategy in the treatment of lymphedema. Andrographolide, a natural product from Andrographis paniculata, is unknown whether andrographolide promotes lymphangiogenesis to improve secondary lymphedema. By using the murine tail lymphedema model, we demonstrated that andrographolide can reduce the thickness of subcutaneous tissue in the mice's tail and enhance lymphatic drainage. Moreover, immunofluorescence staining showed that the number of capillary lymphatic vessels in the ANDRO25 group was significantly more than that in the ANDRO50 and Model groups. Near-infrared lymphography images showed that highlighted sciatic lymph nodes could be seen in the ANDRO25 and ANDRO50 groups. In vitro, andrographolide could promote the proliferation and migration of LEC. In conclusion, andrographolide enhanced the recovery of lymphatic vessels, and promoted lymphatic drainage in the murine tail lymphedema model by promoting the proliferation of lymphatic endothelial cells, thereby reducing the symptoms of lymphedema. This suggested andrographolide may be used as a potential therapeutic drug or medical food ingredient to help patients with secondary lymphedema.

Copper nanoparticles and silver nanoparticles impair lymphangiogenesis in zebrafish.

Lymphatic system distributes in almost all vertebrate tissues and organs, and plays important roles in the regulation of body fluid balance, lipid absorption and immune monitoring. Although CuNPs or AgNPs accumulation has been reported to be closely associated with delayed hatching and motor dysfunction in zebrafish embryos, their biological effects on lymphangiogenesis remain unknown. In this study, thoracic duct was observed to be partially absent in both CuNPs and AgNPs stressed zebrafish larvae. Specifically, CuNPs stress induced hypermethylation of E2F7/8 binding sites on CCBE1 promoters via their producing ROS, thereby leading to the reduction of binding enrichment of E2F7/8 on CCBE1 promoter and its subsequently reduced expression, then resulting in defective lymphatic vessel formation. Differently, AgNPs stress induced down-regulated CCBE1 expression via down-regulating mRNA and protein levels of E2F7/8 transcription factors, thereby resulting in defective lymphatic vessel formation. This study may be the first to demonstrate that CuNPs and AgNPs damaged lymphangiogenesis during zebrafish embryogenesis, mechanistically, CuNPs epigenetically regulated the expression of lymphangiogenesis regulator CCBE1 via hypermethylating its promoter binding sites of E2F7/8, while AgNPs via regulating E2F7/8 expression. Meanwhile, overexpression of ccbe1 mRNA effectively rescued the lymphangiogenesis defects in both AgNPs and CuNPs stressed larvae, while overexpression of e2f7/8 mRNA effectively rescued the lymphangiogenesis defects in AgNPs rather than CuNPs stressed larvae. The results in this study will shed some light on the safety assessment of nanomaterials applied in medicine and on the ecological security assessments of nanomaterials. Video Abstract.

Impact of Hemostasis on the Lymphatic System in Development and Disease.

Lymphatic vessels form a systemic network that maintains interstitial fluid homeostasis and regulates immune responses and is strictly separated from the circulatory system. During embryonic development, lymphatic endothelial cells originate from blood vascular endothelial cells in the cardinal veins and form lymph sacs. Platelets are critical for separating lymph sacs from the cardinal veins through interactions between CLEC-2 (C-type lectin-like receptor-2) and PDPN (podoplanin) in lymphatic endothelial cells. Therefore, deficiencies of these genes cause blood-filled lymphatic vessels, leading to abnormal lymphatic vessel maturation. The junction between the thoracic duct and the subclavian vein has valves and forms physiological thrombi dependent on CLEC-2/PDPN signaling to prevent blood backflow into the thoracic duct. In addition, platelets regulate lymphangiogenesis and maintain blood/lymphatic separation in pathological conditions, such as wound healing and inflammatory diseases. More recently, it was reported that the entire hemostatic system is involved in lymphangiogenesis. Thus, the hemostatic system plays a crucial role in the establishment, maintenance, and rearrangement of lymphatic networks and contributes to body fluid homeostasis, which suggests that the hemostatic system is a potential target for treating lymphatic disorders. This review comprehensively summarizes the role of the hemostatic system in lymphangiogenesis and lymphatic vessel function and discusses challenges and future perspectives.

The extracellular matrix protein EMILIN-1 impacts on the microenvironment by hampering gastric cancer development and progression.

BACKGROUND: The contribution of the tumor microenvironment and extracellular matrix to the aggressive biology of Gastric Cancer (GC) has been recently characterized; however, the role of EMILIN-1 in this context is unknown. EMILIN-1 is an essential structural element for the maintenance of lymphatic vessel (LV) integrity and displays anti-proliferative properties as demonstrated in skin and colon cancer. Given the key role of LVs in GC progression, the aim of this study was to investigate the role of EMILIN-1 in GC mouse models. METHODS: We used the syngeneic YTN16 cells which were injected subcutaneously and intraperitoneally in genetically modified EMILIN-1 mice. In alternative, carcinogenesis was induced using N-Methyl-N-nitrosourea (MNU). Mouse-derived samples and human biopsies were analyzed by IHC and IF to the possible correlation between EMILIN-1 expression and LV pattern. RESULTS: Transgenic mice developed tumors earlier compared to WT animals. 20 days post-injection tumors developed in EMILIN-1 mutant mice were larger and displayed a significant increase of lymphangiogenesis. Treatment of transgenic mice with MNU associated with an increased number of tumors, exacerbated aggressive lesions and higher levels of LV abnormalities. A significant correlation between the levels of EMILIN-1 and podoplanin was detected also in human samples, confirming the results obtained with the pre-clinical models. CONCLUSIONS: This study demonstrates for the first time that loss of EMILIN-1 in GC leads to lymphatic dysfunction and proliferative advantages that sustain tumorigenesis, and assess the use of our animal model as a valuable tool to verify the fate of GC upon loss of EMILIN-1.

Surgical options for lymphedema after gynecological cancer treatment: current trends and advances.

Lower leg lymphedema is an important complication after gynecological treatment that can severely affect the quality of life of long-term survivors of these malignancies. As a chronic and progressive disease, affected patients will require life-long therapy centered on compression. Although conventional compressive treatments can be effective, they are extremely burdensome and time-consuming for most patients and adherence is challenging. With advances in the field of reconstructive microsurgery, new procedures have been developed in the past decades to help these patients in their continuous care and have been offered at many oncological centers around the world as a first line of treatment. We performed a PubMed search using the Mesh terms 'Lymphedema/surgery' and 'Lower extremity' yielding a total of 508 articles. Of these, 35 articles were included for analysis. Articles that failed to provide a comprehensive analysis of outcomes following surgical treatment, studies examining treatment for upper limb lymphedema, primary lymphedema, or lower extremity lymphedema resulting from non-gynecologic etiologies, and studies that failed to have a minimum of 6 months follow-up were excluded. A comprehensive review of these 35 articles including over 1200 patients demonstrated large variability on the outcomes reported; however, an overall benefit from these procedures was found. Surgical options including lymphovenous anastomosis, vascularized lymph node transfers, and excisional procedures can be performed in patients with lower leg lymphedema, depending on staging and findings in indocyanine green lymphography. Surgical treatment of lymphedema is an effective option that can improve symptoms and quality of life of patients suffering from lymphedema following gynecologic cancers.

Unraveling the lymphatic system in the spinal cord meninges: a critical element in protecting the central nervous system.

The lymphatic vasculature plays a crucial role in fluid clearance and immune responses in peripheral organs by connecting them to distal lymph nodes. Recently, attention has been drawn to the lymphatic vessel network surrounding the brain's border tissue (Aspelund et al. in J Exp Med 212:991-999, 2015. https://doi.org/10.1084/jem.20142290 ; Louveau et al. in Nat Neurosci 21:1380-1391, 2018. https://doi.org/10.1038/s41593-018-0227-9 ), which guides immune cells in mediating protection against tumors (Song et al. in Nature 577:689-694, 2020. https://doi.org/10.1038/s41586-019-1912-x ) and pathogens Li et al. (Nat Neurosci 25:577-587, 2022. https://doi.org/10.1038/s41593-022-01063-z ) while also contributing to autoimmunity (Louveau et al. 2018) and neurodegeneration (Da Mesquita et al. in Nature 560:185-191, 2018. https://doi.org/10.1038/s41586-018-0368-8 ). New studies have highlighted the integral involvement of meningeal lymphatic vessels in neuropathology. However, our limited understanding of spinal cord meningeal lymphatics and immunity hinders efforts to protect and heal the spinal cord from infections, injury, and other immune-mediated diseases. This review aims to provide a comprehensive overview of the state of spinal cord meningeal immunity, highlighting its unique immunologically relevant anatomy, discussing immune cells and lymphatic vasculature, and exploring the potential impact of injuries and inflammatory disorders on this intricate environment.

The emerging roles of long noncoding RNAs in lymphatic vascular development and disease.

Recent advances in RNA sequencing technologies helped uncover what was once uncharted territory in the human genome-the complex and versatile world of long noncoding RNAs (lncRNAs). Previously thought of as merely transcriptional "noise", lncRNAs have now emerged as essential regulators of gene expression networks controlling development, homeostasis and disease progression. The regulatory functions of lncRNAs are broad and diverse, and the underlying molecular mechanisms are highly variable, acting at the transcriptional, post-transcriptional, translational, and post-translational levels. In recent years, evidence has accumulated to support the important role of lncRNAs in the development and functioning of the lymphatic vasculature and associated pathological processes such as tumor-induced lymphangiogenesis and cancer metastasis. In this review, we summarize the current knowledge on the role of lncRNAs in regulating the key genes and pathways involved in lymphatic vascular development and disease. Furthermore, we discuss the potential of lncRNAs as novel therapeutic targets and outline possible strategies for the development of lncRNA-based therapeutics to treat diseases of the lymphatic system.

Contribution of initial lymphatics to oral wound healing after tooth extraction.

Lymphatics are involved in the resolution of inflammation and wound healing, but their role in the oral wound healing process after tooth extraction has never been investigated. We therefore sought to evaluate the healing process following the extraction of maxillary molars in two transgenic mouse models: K14-VEGFR3-Ig mice, which lack initial mucosal lymphatic vessels, and K14-VEGFC mice, which have hyperplastic mucosal lymphatics. Maxillary molars were extracted from both transgenic mouse types and their corresponding wild-type (WT) controls. Mucosal and alveolar bone healing were evaluated. A delayed epithelialization and bone regeneration were observed in K14-VEGFR3-Ig mice compared with their WT littermates. The hampered wound closure was accompanied by decreased levels of epidermal growth factor (EGF) and persistent inflammation, characterized by infiltrates of immune cells and elevated levels of pro-inflammatory markers in the wounds. Hyperplastic mucosal lymphatics did not enhance the healing process after tooth extraction in K14-VEGFC mice. The findings indicate that initial mucosal lymphatics play a major role in the initial phase of the oral wound healing process.

Neurosurgery and the glymphatic system.

The discovery of the glymphatic system has fundamentally altered our comprehension of cerebrospinal fluid transport and the removal of waste from brain metabolism. In the past decade, since its initial characterization, research on the glymphatic system has surged exponentially. Its potential implications for central nervous system disorders have sparked significant interest in the field of neurosurgery. Nonetheless, ongoing discussions and debates persist regarding the concept of the glymphatic system, and our current understanding largely relies on findings from experimental animal studies. This review aims to address several key inquiries: What methodologies exist for evaluating glymphatic function in humans today? What is the current evidence supporting the existence of a human glymphatic system? Can the glymphatic system be considered distinct from the meningeal-lymphatic system? What is the human evidence for glymphatic-meningeal lymphatic system failure in neurosurgical diseases? Existing literature indicates a paucity of techniques available for assessing glymphatic function in humans. Thus far, intrathecal contrast-enhanced magnetic resonance imaging (MRI) has shown the most promising results and have provided evidence for the presence of a glymphatic system in humans, albeit with limitations. It is, however, essential to recognize the interconnection between the glymphatic and meningeal lymphatic systems, as they operate in tandem. There are some human studies demonstrating deteriorations in glymphatic function associated with neurosurgical disorders, enriching our understanding of their pathophysiology. However, the translation of this knowledge into clinical practice is hindered by the constraints of current glymphatic imaging modalities.

Novel erectile analyses revealed augmentable penile Lyve-1, the lymphatic marker, expression.

Purpose: The pathophysiology of penis extends to erectile dysfunction (ED) to conditions including sexually transmitted diseases (STDs) and cancer. To date, there has been little research evaluating vascular drainage from the penis. We aimed to evaluate penile blood flow in vivo and analyze its possible relationship with the lymphatic maker. Materials and Methods: We established an in vivo system designed to assess the dynamic blood outflow from the corpus cavernosum (CC) by dye injection. To analyze lymphatic characteristics in the CC, the expression of Lyve-1, the key lymphatic endothelium marker, was examined by the in vitro system and lipopolysaccharide (LPS) injection to mimic the inflammatory conditions. Results: A novel cavernography methods enable high-resolution morphological and functional blood drainage analysis. The expression of Lyve-1 was detected along the sinusoids. Furthermore, its prominent expression was also observed after penile LPS injection and in the erectile condition. Conclusions: The current in vivo system will potentially contribute to the assessment of penile pathology from a novel viewpoint. In addition, current analyses revealed inducible Lyve-1 expression for LPS injection and the erection state, which requires further analyses on penile lymphatic system.

Hyaluronic Acid as a LYVE-1 Receptor Ligand in the Lymphatic System of Healthy Human Skin.

Immunohistochemical detection of the LYVE-1 marker in healthy human full-thickness skin (the epidermis and the dermis) was carried out. LYVE-1 expression was found in the endothelium of lymphatic capillaries located in the papillary dermis, in the endothelium of larger lymphatic vessels of the reticular dermis, and in fibroblasts, which indicates their joint participation in hyaluronan metabolism. LYVE-1+ staining detected for the first time in cells of the stratum basale, the stratum spinosum, and the stratum granulosum of healthy human epidermis indicates their participation in hyaluronan metabolism and allows us to consider the spaces between epidermis cells as prelimphatics.