Lymphatic Vessel Function Research Articles

Current Status of Lymphangiogenesis: Molecular Mechanism, Immune Tolerance, and Application Prospect

The lymphatic system is a channel for fluid transport and cell migration, but it has always been controversial in promoting and suppressing cancer. VEGFC/VEGFR3 signaling has long been recognized as a major molecular driver of lymphangiogenesis. However, many studies have shown that the neural network of lymphatic signaling is complex. Lymphatic vessels have been found to play an essential role in the immune regulation of tumor metastasis and cardiac repair. This review describes the effects of lipid metabolism, extracellular vesicles, and flow shear forces on lymphangiogenesis. Moreover, the pro-tumor immune tolerance function of lymphatic vessels is discussed, and the tasks of meningeal lymphatic vessels and cardiac lymphatic vessels in diseases are further discussed. Finally, the value of conversion therapy targeting the lymphatic system is introduced from the perspective of immunotherapy and pro-lymphatic biomaterials for lymphangiogenesis.

Lymphatic flow velocity is a predictor of functional lymphatic vessels for lymphaticovenous anastomosis

Indocyanine green (ICG) lymphography is widely used to localize functional lymphatic vessels for lymphaticovenous anastomosis (LVA); however, flow velocity is rarely assessed. We aimed to evaluate the correlation between lymphatic flow velocity and the presence of functional lymphatic vessels. Data of a total of 924 lymphatic vessels from 273 lymphedema patients who underwent LVA between July 2018 and December 2020 were retrospectively reviewed. Lymph flow velocity was defined by considering the most proximal anatomic location enhanced by ICG at 30 min after injection and categorized into four groups; grade 1 (foot or hand), grade 2 (below knee or elbow), grade 3 (at/above knee or eblow), or grade 4 (axilla or groin). The presence of functional lymphatic vessels, which showed lymphatic fluid flow when the vessels were cut for anastomosis, was compared between the four groups. A higher rate of functional lymphatic vessels was observed among lymphatic vessels with grade 3 or 4 flow velocity compared with those with grade 1 or 2 flow velocity (67.5% vs. 44.5%; p < 0.001). These findings were consistent with the observations for lymphatic vessels with a non-linear pattern in ICG lymphography (59.4% vs. 26.5%; p < 0.001). The rate of completion of LVA at surgical sites in extremities with grade 3 or 4 flow velocity was 88.1% compared with 65.8% in extremities with grade 1 or 2 velocity (p < 0.001). Lymph flow velocity grading can be a simple and easy adjunctive method to determine indication for LVA in extremities with lymphedema.

Cellular Senescence as a Brake or Accelerator for Oncogenic Transformation and Role in Lymphatic Metastasis.

Cellular senescence-the irreversible cell cycle arrest driven by a variety of mechanisms and, more specifically, the senescence-associated secretory phenotype (SASP)-is an important area of research in the context of different age-related diseases, such as cardiovascular disease and cancer. SASP factors play both beneficial and detrimental roles in age-related disease progression depending on the source of the SASPs, the target cells, and the microenvironment. The impact of senescence and the SASP on different cell types, the immune system, and the vascular system has been widely discussed. However, the impact of replicative or stress-induced senescence on lymphatic biology and pathological lymphangiogenesis remains underexplored. The lymphatic system plays a crucial role in the maintenance of body fluid homeostasis and immune surveillance. The perturbation of lymphatic function can hamper normal physiological function. Natural aging or stress-induced premature aging influences the lymphatic vessel structure and function, which significantly affect the role of lymphatics in tumor dissemination and metastasis. In this review, we focus on the role of senescence on lymphatic pathobiology, its impact on cancer, and potential therapeutic interventions to manipulate the aged or senescent lymphatic system for disease management.

Promotion of Lymphangiogenesis by Targeted Delivery of VEGF-C Improves Diabetic Wound Healing.

Chronic wounds represent a major therapeutic challenge. Lymphatic vessel function is impaired in chronic ulcers but the role of lymphangiogenesis in wound healing has remained unclear. We found that lymphatic vessels are largely absent from chronic human wounds as evaluated in patient biopsies. Excisional wound healing studies were conducted using transgenic mice with or without an increased number of cutaneous lymphatic vessels, as well as antibody-mediated inhibition of lymphangiogenesis. We found that a lack of lymphatic vessels mediated a proinflammatory wound microenvironment and delayed wound closure, and that the VEGF-C/VEGFR3 signaling axis is required for wound lymphangiogenesis. Treatment of diabetic mice (db/db mice) with the F8-VEGF-C fusion protein that targets the alternatively spliced extra domain A (EDA) of fibronectin, expressed in remodeling tissue, promoted wound healing, and potently induced wound lymphangiogenesis. The treatment also reduced tissue inflammation and exerted beneficial effects on the wound microenvironment, including myofibroblast density and collagen deposition. These findings indicate that activating the lymphatic vasculature might represent a new therapeutic strategy for treating chronic non-healing wounds.

Fibroblast-Generated Extracellular Matrix Guides Anastomosis during Wound Healing in an Engineered Lymphatic Skin Flap.

A healthy lymphatic system is required to return excess interstitial fluid back to the venous circulation. However, up to 49% of breast cancer survivors eventually develop breast cancer-related lymphedema due to lymphatic injuries from lymph node dissections or biopsies performed to treat cancer. While early-stage lymphedema can be ameliorated by manual lymph drainage, no cure exists for late-stage lymphedema when lymph vessels become completely dysfunctional. A viable late-stage treatment is the autotransplantation of functional lymphatic vessels. Here we report on a novel engineered lymphatic flap that may eventually replace the skin flaps used in vascularized lymph vessel transfers. The engineered flap mimics the lymphatic and dermal compartments of the skin by guiding multi-layered tissue organization of mesenchymal stem cells and lymphatic endothelial cells with an aligned decellularized fibroblast matrix. The construct was tested in a novel bilayered wound healing model and implanted into athymic nude rats. The in vitro model demonstrated capillary invasion into the wound gaps and deposition of extracellular matrix fibers, which may guide anastomosis and vascular integration of the graft during wound healing. The construct successfully anastomosed in vivo, forming chimeric vessels of human and rat cells. Overall, our flap replacement has high potential for treating lymphedema.

Multiple aspects of lymphatic dysfunction in an ApoE -/- mouse model of hypercholesterolemia.

Introduction: Rodent models of cardiovascular disease have uncovered various types of lymphatic vessel dysfunction that occur in association with atherosclerosis, type II diabetes and obesity. Previously, we presented in vivo evidence for impaired lymphatic drainage in apolipoprotein E null (ApoE -/- ) mice fed a high fat diet (HFD). Whether this impairment relates to the dysfunction of collecting lymphatics remains an open question. The ApoE -/- mouse is a well-established model of cardiovascular disease, in which a diet rich in fat and cholesterol on an ApoE deficient background accelerates the development of hypercholesteremia, atherosclerotic plaques and inflammation of the skin and other tissues. Here, we investigated various aspects of lymphatic function using ex vivo tests of collecting lymphatic vessels from ApoE +/+ or ApoE -/- mice fed a HFD. Methods: Popliteal collectors were excised from either strain and studied under defined conditions in which we could quantify changes in lymphatic contractile strength, lymph pump output, secondary valve function, and collecting vessel permeability. Results: Our results show that all these aspects of lymphatic vessel function are altered in deleterious ways in this model of hypercholesterolemia. Discussion: These findings extend previous in vivo observations suggesting significant dysfunction of lymphatic endothelial cells and smooth muscle cells from collecting vessels in association with a HFD on an ApoE-deficient background. An implication of our study is that collecting vessel dysfunction in this context may negatively impact the removal of cholesterol by the lymphatic system from the skin and the arterial wall and thereby exacerbate the progression and/or severity of atherosclerosis and associated inflammation.

The lymphatic system: a therapeutic target for central nervous system disorders.

The lymphatic vasculature forms an organized network that covers the whole body and is involved in fluid homeostasis, metabolite clearance, and immune surveillance. The recent identification of functional lymphatic vessels in the meninges of the brain and the spinal cord has provided novel insights into neurophysiology. They emerge as major pathways for fluid exchange. The abundance of immune cells in lymphatic vessels and meninges also suggests that lymphatic vessels are actively involved in neuroimmunity. The lymphatic system, through its role in the clearance of neurotoxic proteins, autoimmune cell infiltration, and the transmission of pro-inflammatory signals, participates in the pathogenesis of a variety of neurological disorders, including neurodegenerative and neuroinflammatory diseases and traumatic injury. Vascular endothelial growth factor C is the master regulator of lymphangiogenesis, a process that is critical for the maintenance of central nervous system homeostasis. In this review, we summarize current knowledge and recent advances relating to the anatomical features and immunological functions of the lymphatic system of the central nervous system and highlight its potential as a therapeutic target for neurological disorders and central nervous system repair.

Networked lymphatic endothelial cells in a transplanted cell sheet contribute to form functional lymphatic vessels

This study evaluated whether cell sheets containing a network of lymphatic endothelial cells (LECs) promoted lymphangiogenesis after transplantation in vivo. Cell sheets with a LEC network were constructed by co-culturing LECs and adipose-derived stem cells (ASCs) on temperature-responsive culture dishes. A cell ratio of 3:2 (vs. 1:4) generated networks with more branches and longer branch lengths. LEC-derived lymphatic vessels were observed 2 weeks after transplantation of a three-layered cell sheet construct onto rat gluteal muscle. Lymphatic vessel number, diameter and depth were greatest for a construct comprising two ASC sheets stacked on a LEC/ASC (3:2 ratio) sheet. Transplantation of this construct in a rat model of femoral lymphangiectomy led to the formation of functional lymphatic vessels containing both transplanted and host LECs. Further development of this technique may lead to a new method of promoting lymphangiogenesis.

Photodynamic Opening of the Blood-Brain Barrier and the Meningeal Lymphatic System: The New Niche in Immunotherapy for Brain Tumors.

Photodynamic therapy (PDT) is a promising add-on therapy to the current standard of care for patients with glioblastoma (GBM). The traditional explanation of the anti-cancer PDT effects involves the PDT-induced generation of a singlet oxygen in the GBM cells, which causes tumor cell death and microvasculature collapse. Recently, new vascular mechanisms of PDT associated with opening of the blood-brain barrier (OBBB) and the activation of functions of the meningeal lymphatic vessels have been discovered. In this review, we highlight the emerging trends and future promises of immunotherapy for brain tumors and discuss PDT-OBBB as a new niche and an important informative platform for the development of innovative pharmacological strategies for the modulation of brain tumor immunity and the improvement of immunotherapy for GBM.

Lentiviral overexpression of VEGFC in transplanted MSCs leads to resolution of swelling in a mouse tail lymphedema model.

Dysfunction of the lymphatic system following injury, disease, or cancer treatment can lead to lymphedema, a debilitating condition with no cure. Despite the various physical therapy and surgical options available, most treatments are palliative and fail to address the underlying lymphatic vascular insufficiency driving lymphedema progression. Stem cell therapy provides a promising alternative in the treatment of various chronic diseases with a wide range of therapeutic effects that reduce inflammation, fibrosis, and oxidative stress, while promoting lymphatic vessel (LV) regeneration. Specifically, stem cell transplantation is suggested to promote LV restoration, rebuild lymphatic circulation, and thus potentially be utilized towards an effective lymphedema treatment. In addition to stem cells, studies have proposed the administration of vascular endothelial growth factor C (VEGFC) to promote lymphangiogenesis and decrease swelling in lymphedema. Here, we seek to combine the benefits of stem cell therapy, which provides a cellular therapeutic approach that can respond to the tissue environment, and VEGFC administration to restore lymphatic drainage. Specifically, we engineered mesenchymal stem cells (MSCs) to overexpress VEGFC using a lentiviral vector (hVEGFC MSC) and investigated their therapeutic efficacy in improving LV function and tissue swelling using near infrared (NIR) imaging, and lymphatic regeneration in a single LV ligation mouse tail lymphedema model. First, we showed that overexpression of VEGFC using lentiviral transduction led to an increase in VEGFC protein synthesis in vitro. Then, we demonstrated hVEGFC MSC administration post-injury significantly increased the lymphatic contraction frequency 14-, 21-, and 28-days post-surgery compared to the control animals (MSC administration) in vivo, while also reducing tail swelling 28-days post-surgery compared to controls. Our results suggest a therapeutic potential of hVEGFC MSC in alleviating the lymphatic dysfunction observed during lymphedema progression after secondary injury and could provide a promising approach to enhancing autologous cell therapy for treating lymphedema.

Improving the Function of Meningeal Lymphatic Vessels to Promote Brain Edema Absorption after Traumatic Brain Injury.

Brain edema is the most common and fatal complication after traumatic brain injury (TBI). Meningeal lymphatic vessels (MLVs) are the conduits that transport cerebrospinal fluid (CSF) and macromolecules to deep extracranial cervical lymph nodes (dCLNs). After TBI, the drainage function of MLVs can become impaired. However, the scenario in which the improvement of the function of MLVs can promote brain edema absorption after TBI has not been reported. Therefore, the purpose of this study was to investigate the effects of ketoprofen, 9-cis retinoic acid (RA) and vascular endothelial cell growth factor-C (VEGF-C), which promote the proliferation of peripheral lymphatic vessels, on the cerebellar medullary cistern injection of TBI rats, as well as their mechanism of action on brain edema after TBI. In the experiment, we found that ketoprofen, 9-cisRA, and VEGF-C can improve the function of MLVs, promote the extracranial drainage of CSF and the absorption of brain edema, weaken the neuroinflammatory response, reduce reactive oxygen species (ROS) production, maintain the structural integrity of MLVs, and improve neurological function. In addition, ketoprofen, 9-cisRA, and VEGF-C upregulated the lymphatic-specific proteins VEGF receptor (VEGFR)3, PROX1, forkhead box protein C2 (FOXC2), and lymphatic vessel endothelial hyaluronan receptor 1 (LYVE1). These results indicate that ketoprofen, 9-cisRA, and VEGF-C may maintain the integrity of the meningeal lymphatic wall and promote lymphatic proliferation by upregulating the expression of lymphatic vessel-specific proteins, improve meningeal lymphatic function after TBI, promote CSF drainage and brain edema absorption, reduce the immune response of the nervous system, and reduce ROS formation, thereby improving prognoses. These findings may provide new ideas for the treatment of brain edema after TBI.

Vascular endothelial growth factor C gene expression and its serum level as potential biomarkers for obesity in Egyptian children

Background and objectivesObesity is a serious health issue among children, and it's linked to a higher risk of chronic illness. Increased adipose tissue, which is highly vascularized due to angiogenesis, causes adverse health problems. Angiogenesis and lymphangiogenesis are regulated by vascular endothelial growth factor C (VEGF-C), and poor lymphatic vessel function is linked to increased adipocyte accumulation. Therefore, our study analyzes VEGF-C gene expression and its serum level in children with obesity in Egypt and correlates them with metabolic, clinical, and biochemical parameters. Subjects & methodsWe compared VEGF-C gene expression in blood and its serum level from 50 unrelated children with obesity and 50 normal healthy controls chosen randomly of matched age and gender recruited from Pediatric Clinics, both inpatient and outpatient, Menoufia University, Egypt, from March to June 2021, in a case-control study. The expression levels of VEGF-C mRNA were evaluated using a real-time quantitative reverse transcription-polymerase chain reaction and the serum VEGF-C level was measured using an enzyme-linked immunosorbent assay (ELISA). ResultsFasting plasma glucose, fasting insulin, homeostatic model assessment for insulin resistance (HOMA-IR), triacylglycerol (TAG), total cholesterol, low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDLC), serum VEGF-C, and VEGF-C expression levels were significantly higher in obese children group compared with the control (P < 0.001). In addition, serum VEGF-C levels were significantly inversely correlated with FT4 (P = 0.009) and significantly directly correlated with total cholesterol (P = 0.005) and LDL-C levels (P = 0.006) in the obese children group. Moreover, VEGF-C expression levels were also significantly inversely correlated with HDL-C levels (P = 0.001) and significantly directly correlated with fasting insulin (P = 0.002) and HOMA-IR (P = 0.001) in the obese children group. ConclusionVEGF-C gene expression and its serum levels could be used for follow-up of obese children with metabolic abnormalities.

Investigations on the donor limb after harvest of lymphatic vessels for lymphedema surgery

ObjectiveMicrosurgical lymphatic vessel transplantation is one of the well-established therapies for lymphedema. Lymphatic vessels are harvested from a healthy thigh and transplanted into lymphedematous limbs to create a lymphatic bypass. Its benefit on lymphatic drainage has already been proven. However, to the best of our knowledge, the effect on the lymphatic function of the donor site has not yet been studied. Our aim was to evaluate the long-term postoperative lymphatic function in the donor site by clinical and scintigraphic examinations and a patient questionnaire. MethodsA consecutive series of 25 women (mean age, 57.2 years) who had undergone follow-up after lymph vessel transplantation to treat secondary lymphedema of the arm comprised the study group. Lymphatic vessel function of the donor site was evaluated by circumferential measurements of the limb and, in nine cases, by lymphatic scintigraphy. Additionally, a questionnaire was used to assess the patients’ pre- and postoperative complaints for the donor limb and quality of life. Separately, the medical records of 100 patients who had undergone lymphatic harvest and been followed up were reviewed for documented signs of lymphatic function of the donor limb. ResultsThe lymphatic grafts were harvested from the thigh (left, n = 9; right, n = 16) and transplanted to bridge the region of lymphatic obstruction in the axilla. The mean follow-up period was 4.5 years after surgery. None of the patients had shown significant changes in the circumference of the donor limb or pathologic findings via lymphatic scintigraphy. None of the patients had reported any impairment in the donor leg or showed symptoms of postoperative lymphedema or erysipelas. ConclusionsOur results have shown that harvesting lymphatic vessels from the thigh for lymphatic vessel transplantation is possible without significant donor site morbidity.

Persistent popliteal lymphatic muscle cell coverage defects despite amelioration of arthritis and recovery of popliteal lymphatic vessel function in TNF-Tg mice following anti-TNF therapy

While rheumatoid arthritis patients and tumor necrosis factor transgenic (TNF-Tg) mice with inflammatory-erosive arthritis display lymphatic drainage deficits, the mechanisms responsible remain unknown. As ultrastructural studies of joint-draining popliteal lymphatic vessels (PLVs) in TNF-Tg mice revealed evidence of lymphatic muscle cell (LMC) damage, we aimed to evaluate PLV-LMC coverage in TNF-Tg mice. We tested the hypothesis that alpha smooth muscle actin (αSMA)+ PLV-LMC coverage decreases with severe inflammatory-erosive arthritis, and is recovered by anti-TNF therapy facilitated by increased PLV-LMC turnover during amelioration of joint disease. TNF-Tg mice with established disease received anti-TNF monoclonal antibody (mAb) or placebo IgG isotype control mAb therapy (n = 5) for 6-weeks, while wild-type (WT) littermates (n = 8) received vehicle (PBS). Bromodeoxyuridine (BrdU) was also administered daily during the treatment period to monitor PLV-LMC turnover. Effective anti-TNF therapy was confirmed by longitudinal assessment of popliteal lymph node (PLN) volume via ultrasound, PLV contraction frequency via near-infrared imaging of indocyanine green, and ankle bone volumes via micro-computed tomography (micro-CT). Terminal knee micro-CT, and ankle and knee histology were also performed. PLVs were immunostained for αSMA and BrdU to evaluate PLV-LMC coverage and turnover, respectively, via whole-mount fluorescent microscopy. Anti-TNF therapy reduced PLN volume, increased talus and patella bone volumes, and reduced tarsal and knee synovial areas compared to placebo treated TNF-Tg mice (p < 0.05), as expected. Anti-TNF therapy also increased PLV contraction frequency at 3-weeks (from 0.81 ± 1.0 to 3.2 ± 2.0 contractions per minute, p < 0.05). However, both anti-TNF and placebo treated TNF-Tg mice exhibited significantly reduced αSMA+ PLV-LMC coverage compared to WT (p < 0.05). There was no correlation of αSMA+ PLV-LMC coverage restoration with amelioration of inflammatory-erosive arthritis. Similarly, there was no difference in PLV-LMC turnover measured by BrdU labeling between WT, TNF-Tg placebo, and TNF-Tg anti-TNF groups with an average of < 1% BrdU+ PLV-LMCs incorporated per week. Taken together these results demonstrate that PLV-LMC turnover in adult mice is limited, and that recovery of PLV function during amelioration of inflammatory-erosive arthritis occurs without restoration of αSMA+ LMC coverage. Future studies are warranted to investigate the direct and indirect effects of chronic TNF exposure, and the role of proximal inflammatory cells on PLV contractility.

EMILIN-1 deficiency promotes chronic inflammatory disease through TGFβ signaling alteration and impairment of the gC1q/α4β1 integrin interaction

Alterations in extracellular matrix (ECM) components that modulate inflammatory cell behavior have been shown to serve as early starters for multifactorial diseases such as fibrosis and cancer. Here, we demonstrated that loss of the ECM glycoprotein EMILIN-1 alters the inflammatory context in skin during IMQ-induced psoriasis, a disease characterized by a prominent inflammatory infiltrate and alteration of vessels that appear dilated and tortuous. Abrogation of EMILIN-1 expression or expression of the EMILIN-1 mutant E933A impairs macrophage polarization and leads to imbalanced tissue homeostasis. We found that EMILIN-1 deficiency is associated with dilated lymphatic vessels, increased macrophage recruitment and psoriasis severity. Importantly, the null or mutant EMILIN-1 background was characterized by the induction of a myofibroblast phenotype, which in turn drove macrophages towards the M1 phenotype. By using the transgenic mouse model carrying the E933A mutation in the gC1q domain of EMILIN-1, which abolishes the interaction with α4- and α9-integrins, we demonstrated that the observed changes in TGFβ signaling were due to both the EMI and gC1q domains of EMILIN-1. gC1q may exert multiple functions in psoriasis, in the context of a final, more consistent inflammatory condition by controlling skin homeostasis via interaction with both keratinocytes and fibroblasts, influencing non-canonical TGFβ signaling, and likely acting on lymphatic vessel structure and function. The analyses of human psoriatic lesions, in which lower levels of EMILIN-1 were present with a very rare association with lymphatic vessels, support the multifaceted role of this ECM component in the skin inflammatory scenario.

Lymphatic function and anatomy in early stages of lipedema.

ObjectiveLipedema is an inflammatory subcutaneous adipose tissue disease that develops in women and may progress to lipolymphedema, a condition similar to lymphedema, in which lymphatic dysfunction results in irresolvable edema. Because it has been shown that dilated lymphatic vessels, impaired pumping, and dermal backflow are associated with presymptomatic, cancer‐acquired lymphedema, this study sought to understand whether these abnormal lymphatic characteristics also characterize early stages of lipedema prior to lipolymphedema development.MethodsIn a pilot study of 20 individuals with Stage I or II lipedema who had not progressed to lipolymphedema, lymphatic vessel anatomy and function in upper and lower extremities were assessed by near‐infrared fluorescence lymphatic imaging and compared with that of a control population of similar age and BMI.ResultsThese studies showed that, although lower extremity lymphatic vessels were dilated and showed intravascular pooling, the propulsion rates significantly exceeded those of control individuals. Upper extremity lymphatics of individuals with lipedema were unremarkable. In contrast to individuals with lymphedema, individuals with Stage I and II lipedema did not exhibit dermal backflow.ConclusionsThese results suggest that, despite the confusion in the diagnoses between lymphedema and lipedema, their etiologies differ, with lipedema associated with lymphatic vessel dilation but not lymphatic dysfunction.

Receptor Activity Modifying Protein RAMP Sub-Isoforms and Their Functional Differentiation, Which Regulates Functional Diversity of Adrenomedullin.

Simple SummaryThe body contains a variety of humoral bioactive factors and information regulation systems that maintain homeostasis. Although adrenomedullin (AM) was first identified as a vasodilator peptide, subsequent studies have revealed that it plays an essential role in homeostasis in vivo and possesses a variety of bioactivities other than vasodilation. Based on its structural homology, AM is classified as a member of the calcitonin superfamily, which includes calcitonin, calcitonin gene-related peptide (CGRP), amylin, and intermedin. Peptides belonging to the calcitonin superfamily are characterized by a partial sharing of their receptor system. One of the sub-isoforms of receptor activity-modifying protein (RAMP) binds to calcitonin receptor-like receptor (CLR), a 7-transmembrane G protein, in a one-to-one manner, and then functions as a receptor for these peptides. Among RAMP isoforms, we focused on RAMP2 and RAMP3. Each RAMP isoform is expected to be a potential therapeutic target for various diseases, including cardiovascular diseases and cancer metastasis. In this review, we outline the pathophysiological significance and functional differentiation of RAMP2 and RAMP3, as mainly revealed by the analysis of genetically engineered mice.AM knockout (AM-/-) and RAMP2 knockout (RAMP2-/-) proved lethal for mice due to impaired embryonic vascular development. Although most vascular endothelial cell-specific RAMP2 knockout (E-RAMP2-/-) mice also died during the perinatal period, a few E-RAMP2-/- mice reached adulthood. Adult E-RAMP2-/- mice developed spontaneous organ damage associated with vascular injury. In contrast, adult RAMP3 knockout (RAMP3-/-) mice showed exacerbated postoperative lymphedema with abnormal lymphatic drainage. Thus, RAMP2 is essential for vascular development and homeostasis and RAMP3 is essential for lymphatic vessel function. Cardiac myocyte-specific RAMP2 knockout mice showed early onset of heart failure as well as abnormal mitochondrial morphology and function, whereas RAMP3-/- mice exhibited abnormal cardiac lymphatics and a delayed onset of heart failure. Thus, RAMP2 is essential for maintaining cardiac mitochondrial function, while RAMP3 is essential for cardiac lymphangiogenesis. Transplantation of cancer cells into drug-inducible vascular endothelial cell-specific RAMP2 knockout mice resulted in enhanced metastasis to distant organs, whereas metastasis was suppressed in RAMP3-/- mice. RAMP2 suppresses cancer metastasis by maintaining vascular homeostasis and inhibiting vascular inflammation and pre-metastatic niche formation, while RAMP3 promotes cancer metastasis via malignant transformation of cancer-associated fibroblasts. Focusing on the diverse physiological functions of AM and the functional differentiation of RAMP2 and RAMP3 may lead to the development of novel therapeutic strategies.

Assessment of the lymphatic system by indirect lymphography in patients with post-thrombotic syndrome

ObjectiveAlterations in lower extremity lymph drainage caused by chronic venous obstruction (CVO) have not been well studied, partially because of a lack of standardized imaging modalities to assess the quality of lymphatic drainage in the lower extremities of patients with post-thrombotic syndrome (PTS). However, these changes are likely to have an impact on the severity of the disease and clinical outcomes of interventions. In the present study, we investigated the feasibility and diagnostic value of preintervention indirect lymphography in patients with CVO and their pre- and postintervention Villalta scores. MethodsA total of 17 patients (21 limbs) with iliofemoral and caval CVO were included in the study between 2017 and 2018. The deep and superficial lymphatic vessels in both legs were assessed before venous recanalization and stenting. The quality of lymphatic flow was compared between the legs with CVO and healthy legs. Moreover, the correlation between the lymphatic changes and clinical severity of PTS was evaluated using the Villalta score and CEAP (Clinical, Etiology, Anatomy, and Pathophysiology) classification. ResultsThe mean patient age was 44 ± 12 years, and 10 patients (59%) were women. The patients had undergone treatment at a mean of 25 ± 6 months after their first episode of deep vein thrombosis. Five patients (29%) had had recurrent deep vein thrombosis. The mean pre- and postinterventional Villalta score was 10.5 ± 1.46 and 9.27 ± 1.12, respectively (P = .0096). Using the CEAP classification, four legs were class 5, seven were class 4, and three each were class 3 and 2. The primary and secondary patency rate was 70.5% and 82.5% after a mean follow-up of 18 months, respectively. Indirect lymphography of the superficial and deep lymphatic systems was completed before intervention in both legs for all 17 patients (21 legs). According to the qualitative criteria, abnormal lymphatic vessel function was found in 35.2% of the superficial and 58.8% of the deep lymphatic vessels of the affected legs. Further analysis revealed abnormal function of the deep lymphatic vessels in all patients with moderate to severe PTS according to the Villalta score. ConclusionsIndirect lymphography is a feasible diagnostic tool to use for the evaluation of the function of lymphatic vessels. Impaired drainage of the deep lymphatic system was found in all our patients with moderate to severe PTS. The clinical significance of these lymphatic changes is not clear; however, an association between clinical severity and outcomes is possible.

Endothelial Microparticle-Mediated Transfer of microRNA-19b Inhibits the Function and Distribution of Lymphatic Vessels in Atherosclerotic Mice

In recent years, the function of the lymphatic system in atherosclerosis has attracted attention due to its role in immune cell trafficking, cholesterol removal from the periphery, and regulation of the inflammatory response. However, knowledge of the mechanisms regulating lymphangiogenesis and lymphatic function in the pathogenesis of atherosclerosis is limited. Endothelial microparticles carrying circulating microRNA (miRNA)s are known to mediate cell–cell communication, and our previous research showed that miRNA-19b in EMPs (EMPmiR-19b) was significantly increased in circulation and atherosclerotic vessels, and this increase in EMPmiR-19b promoted atherosclerosis. The present study investigated whether atherogenic EMPmiR-19b influences pathological changes of the lymphatic system in atherosclerosis. We first verified increased miR-19b levels and loss of lymphatic system function in atherosclerotic mice. Atherogenic western diet-fed ApoE-/- mice were injected with phosphate-buffered saline, EMPs carrying control miRNA (EMPcontrol), or EMPmiR-19b intravenously. The function and distribution of the lymphatic system was assessed via confocal microscopy, Evans blue staining, and pathological analysis. The results showed that lymphatic system dysfunction existed in the early stage of atherosclerosis, and the observed pathological changes persisted at the later stage, companied by an increased microRNA-19b level. In ApoE-/- mice systemically treated with EMPmiR-19b, the distribution, transport function, and permeability of the lymphatic system were significantly inhibited. In vitro experiments showed that miRNA-19b may damage the lymphatic system by inhibiting lymphatic endothelial cell migration and tube formation, and a possible mechanism is the inhibition of transforming growth factor beta receptor type II (TGF-βRII) expression in lymphatic endothelial cells by miRNA-19b. Together, our findings demonstrate that atherogenic EMPmiR-19b may destroy lymphatic system function in atherosclerotic mice by downregulating TGF-βRII expression.

Effects of Acetate on Mesenteric Lymphatic Pump Function

Pump function of mesenteric lymphatic vessels (MLVs) is essential for active transport of intestinal lymph and lipids from the gut to the venous circulation. MLV pump function is also critical for transport of immune cells in the GI tract, which account for almost 70% of the body’s immune cells. Short‐chain fatty acids (SCFAs)—the metabolites of the intestinal microbiota—are absorbed rapidly across the intestinal epithelial barrier and their concentration reaches 1‐100 mM in the intestinal tissues. There is a growing interest in the effects SCFAs on blood vessels. Blood vessel studies using acetate, the most abundant SCFA in peripheral circulation, reported that vasodilation induced by acetate mediates the blood pressure lowering effects of acetate. Furthermore, nitric oxide (NO) pathway was identified to mediate the acetate‐induced vasodilation. MLVs are exposed to significantly higher SCFA concentrations (as high as those in the intestinal tissues) in the undiluted lymph compared to the blood vessels. However, the effects of SCFAs on lymphatic vessel function have yet to be investigated thoroughly. Therefore, the purpose of the present study was to evaluate our hypothesis that acetate induces lymphatic dilation and decreases lymphatic pump function.MLVs were isolated from male Sprague Dawley rats and cannulated and perfused with warm APSS. Transmural pressure was set to 3 cmH2O and MLVs were allowed to equilibrate. In spontaneously contracting vessels, changes in pump function in response to cumulative concentrations of acetate (10‐8 to 10‐2M) were determined. In separate MLVs, pump function at three transmural pressures (3, 5 and 7 cmH2O; selected randomly) was evaluated before and after incubating the MLVs with acetate (10 mM). Additionally, in another set of MLVs, lymphatic pump function at three transmural pressures (3, 5 and 7 cmH2O; selected randomly) was evaluated before and after incubating the MLVs with N(ω)‐nitro‐L‐arginine methyl ester (L‐NAME; 100 µM) and again after incubating the MLVs with L‐NAME (100 µM) + acetate (10 mM).The dose‐response study revealed that acetate led to decrease in lymphatic contraction frequency and calculated active lymph flow in a dose‐dependent manner. IC50 of the dose‐response curve was 1mM and lymphatic pumping was completely abolished at 50 mM concentration. Lymphatic contraction frequency and calculated active lymph flow were significantly decreased at all transmural pressures. Lymphatic stroke volume normalized to passive volume and diastolic tone were not significantly altered by acetate. Blockade of the NO pathway with L‐NAME did not restore the acetate‐induced decrease in lymphatic contraction frequency at all transmural pressures.Consistent with the effects of acetate on the blood vessels, acetate decreased lymphatic pump function in a dose‐dependent manner. However, the findings suggest that the NO pathway, reported to mediate blood vessel responses to acetate, does not mediate lymphatic responses to acetate. Two different pathways mediating acetate‐induced vasodilation in blood and lymphatic vessels may enable regulation of blood or lymphatic vessel function independently.