Lymphatic vessel transit seeds cytotoxic resident memory T cells in skin draining lymph nodes.

Lymphatic transport shapes the homeostatic immune repertoire of lymph nodes (LNs). LN-resident memory T cells (TRMs) play an important role in site-specific immune memory, yet how LN TRMs form de novo after viral infection remains unclear. Here, we tracked the anatomical distribution of antiviral CD8+ T cells as they seeded skin and LN TRMs using a model of vaccinia virus-induced skin infection. LN TRMs localized to the draining LNs (dLNs) of infected skin, and their formation depended on the lymphatic egress of effector CD8+ T cells from the skin, already poised for residence. Effector CD8+ T cell transit through skin was required to populate LN TRMs in dLNs, a process reinforced by antigen encounter in skin. Furthermore, LN TRMs were protective against viral rechallenge in the absence of circulating memory T cells. These data suggest that a subset of tissue-infiltrating CD8+ T cells egress from tissues during viral clearance and establish a layer of regional protection in the dLN basin.

Analysis on efficacy of magnetic resonance lymphangiography using INV-001 in healthy beagle dogs.

We aimed to conduct a proof-of-concept study of INV-001 in visualizing lymphatic vessels and nodes without venous contamination and to determine the optimal dose condition of INV-001 for magnetic resonance lymphangiography (MRL) in healthy beagles. MRL was performed using a 3.0-Tesla (T) whole body clinical magnetic resonance imaging (MRI) scanner. A dose-finding study of INV-001 for MRL in beagles (N = 6) was carried out according to an adaptive optimal dose finding design. For the reproducibility study (N = 6), MRL was conducted at selected INV-001 doses (0.056 and 0.112 mg Fe/kg) with a 15 mM concentration. Additionally, an excretion study (N = 3) of INV-001 was conducted by analyzing T1, T2, and T2* maps of the liver and kidney 48 h post-administration. INV-001 administration at doses of 0.056 and 0.112 mg Fe/kg (concentration: 15 mM) consistently demonstrated the visualization of contrast-enhanced lymphatic vessels and nodes without venous contamination in the beagles. The contrast enhancement effect was highest at 30 min after INV-001 administration, then gradually decreasing. No toxicity-related issues were identified during the study. After 48 h, the T1, T2, and T2* values in the liver and both kidneys were found to be comparable to the pre-administration values, indicating thorough INV-001 excretion. The optimal dosing conditions of INV-001 for MRL for contrast-enhanced visualization of lymphatic vessels and nodes exclusively with no venous contamination in beagles was determined to be 0.056 mg Fe/kg with a 15 mM concentration.

Robot-assisted lymphovenous anastomosis surgery for lymphocele in the groin.

We present the first-in-human robot-assisted microsurgery on a lymphocele in the groin involving a man in his late 60s who had been coping with the condition for 12 months. Despite numerous efforts at conservative treatment and surgical intervention, the lymphocele persisted, leading to a referral to our clinic.Diagnostic techniques, including indocyanine green lymphography and ultrasound, identified one lymphatic vessel draining into the lymphocele. The surgical intervention, conducted with the assistance of a robot and facilitated by the Symani Surgical System (Medical Microinstruments, Calci, Italy), involved a lymphovenous anastomosis and excision of the lymphocele. An end-to-end anastomosis was performed between the lymphatic and venous vessels measuring 1 mm in diameter, using an Ethilon 10-0 suture.The surgery was successful, with no postoperative complications and a prompt recovery. The patient was discharged 3 days postoperatively and exhibited complete recovery at the 14-day follow-up. This case marks the first use of robot-assisted microsurgical lymphovenous anastomosis to address a groin lymphocele, highlighting the benefit of advanced robotic technology in complex lymphatic surgeries.

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.

Multiple cis-regulatory elements control prox1a expression in distinct lymphatic vascular beds.

During embryonic development, lymphatic endothelial cell (LEC) precursors are distinguished from blood endothelial cells by the expression of Prospero-related homeobox 1 (Prox1), which is essential for lymphatic vasculature formation in mouse and zebrafish. Prox1 expression initiation precedes LEC sprouting and migration, serving as the marker of specified LECs. Despite its crucial role in lymphatic development, Prox1 upstream regulation in LECs remains to be uncovered. SOX18 and COUP-TFII are thought to regulate Prox1 in mice by binding its promoter region. However, the specific regulation of Prox1 expression in LECs remains to be studied in detail. Here, we used evolutionary conservation and chromatin accessibility to identify enhancers located in the proximity of zebrafish prox1a active in developing LECs. We confirmed the functional role of the identified sequences through CRISPR/Cas9 mutagenesis of a lymphatic valve enhancer. The deletion of this region results in impaired valve morphology and function. Overall, our results reveal an intricate control of prox1a expression through a collection of enhancers. Ray-finned fish-specific distal enhancers drive pan-lymphatic expression, whereas vertebrate-conserved proximal enhancers refine expression in functionally distinct subsets of lymphatic endothelium.

Angpt1 binding to Tie1 regulates the signaling required for lymphatic vessel development in zebrafish.

Development of the vascular system is regulated by multiple signaling pathways mediated by receptor tyrosine kinases. Among them, angiopoietin (Ang)/Tie signaling regulates lymphatic and blood vessel development in mammals. Of the two Tie receptors, Tie2 is well known as a key mediator of Ang/Tie signaling, but, unexpectedly, recent studies have revealed that the Tie2 locus has been lost in many vertebrate species, whereas the Tie1 gene is more commonly present. However, Tie1-driven signaling pathways, including ligands and cellular functions, are not well understood. Here, we performed comprehensive mutant analyses of angiopoietins and Tie receptors in zebrafish and found that only angpt1 and tie1 mutants show defects in trunk lymphatic vessel development. Among zebrafish angiopoietins, only Angpt1 binds to Tie1 as a ligand. We indirectly monitored Ang1/Tie1 signaling and detected Tie1 activation in sprouting endothelial cells, where Tie1 inhibits nuclear import of EGFP-Foxo1a. Angpt1/Tie1 signaling functions in endothelial cell migration and proliferation, and in lymphatic specification during early lymphangiogenesis, at least in part by modulating Vegfc/Vegfr3 signaling. Thus, we show that Angpt1/Tie1 signaling constitutes an essential signaling pathway for lymphatic development in zebrafish.

Zmiz1 is a novel regulator of lymphatic endothelial cell gene expression and function.

Zinc Finger MIZ-Type Containing 1 (Zmiz1), also known as ZIMP10 or RAI17, is a transcription cofactor and member of the Protein Inhibitor of Activated STAT (PIAS) family of proteins. Zmiz1 is critical for a variety of biological processes including vascular development. However, its role in the lymphatic vasculature is unknown. In this study, we utilized human dermal lymphatic endothelial cells (HDLECs) and an inducible, lymphatic endothelial cell (LEC)-specific Zmiz1 knockout mouse model to investigate the role of Zmiz1 in LECs. Transcriptional profiling of ZMIZ1-deficient HDLECs revealed downregulation of genes crucial for lymphatic vessel development. Additionally, our findings demonstrated that loss of Zmiz1 results in reduced expression of proliferation and migration genes in HDLECs and reduced proliferation and migration in vitro. We also presented evidence that Zmiz1 regulates Prox1 expression in vitro and in vivo by modulating chromatin accessibility at Prox1 regulatory regions. Furthermore, we observed that loss of Zmiz1 in mesenteric lymphatic vessels significantly reduced valve density. Collectively, our results highlight a novel role of Zmiz1 in LECs and as a transcriptional regulator of Prox1, shedding light on a previously unknown regulatory factor in lymphatic vascular biology.

Lymphaticovenous anastomosis in rabbits: A novel live experimental animal model for supermicrosurgical training.

BACKGROUND: Lymphaticovenous anastomosis is widely used in lymphedema management. Although its effectiveness in reducing edema in patients can be clinically observed, evaluating the long-term outcomes of this technique can be complex. This study established an animal model to assess the outcomes of lymphaticovenous anastomosis technique at 15 and 30-days post-surgery using indocyanine green lymphography, Patent Blue V dye injection, and histopathological examination. METHODS: An experimental model was established in the hindlimbs of 10 rabbits using the popliteal vein and afferent lymphatic vessels in the popliteal area. The subjects were divided into two groups: the first group (n = 5) underwent patency assessment at 0 and 15 days, and the second group (n = 5) at 0 and 30-days, resulting in 20 anastomoses. Patency was verified at 0, 15, and 30-days using indocyanine green lymphography and Patent Blue V injection. Histopathological examinations were performed on the collected anastomosis samples. RESULTS: The patency rate was 90% (19/20) initially, 60% (6/10) at 15 days post-surgery, and 80% (8/10) at 30-days. The average diameter of lymphatic vessels and veins was 1.0 mm and 0.8 mm, respectively. The median number of collateral veins was 3; the median surgical time was 65.8 min. Histopathology revealed minimal endothelial damage and inflammatory responses due to the surgical sutures, with vascular inflammation and thrombosis in a single case. Local vascular neoformations were observed. CONCLUSION: This study highlights the reliability and reproducibility of using rabbits as experimental models for training in lymphaticovenous anastomosis technique owing to the accessibility of the surgical site and dimensions of their popliteal vasculature.

Lymphatic network drainage resolves cerebral edema and facilitates recovery from experimental cerebral malaria.

While brain swelling, associated with fluid accumulation, is a known feature of pediatric cerebral malaria (CM), how fluid and macromolecules are drained from the brain during recovery from CM is unknown. Using the experimental CM (ECM) model, we show that fluid accumulation in the brain during CM is driven by vasogenic edema and not by perivascular cerebrospinal fluid (CSF) influx. We identify that fluid and molecules are removed from the brain extremely quickly in mice with ECM to the deep cervical lymph nodes (dcLNs), predominantly through basal routes and across the cribriform plate and the nasal lymphatics. In agreement, we demonstrate that ligation of the afferent lymphatic vessels draining to the dcLNs significantly impairs fluid drainage from the brain and lowers anti-malarial drug recovery from the ECM syndrome. Collectively, our results provide insight into the pathways that coordinate recovery from CM.

Alternative Lymphatic Drainage Pathways in the Trunk Following Oncologic Therapy.

BACKGROUND: Anatomic and functional descriptions of trunk and breast lymphedema following breast cancer treatment are emerging as indicators of lymphatic dysfunction. Indocyanine green-lymphangiography has been instrumental in characterizing this dysfunction in the extremity and can be applied to other regions. Previous work has established a validated Pittsburgh Trunk Lymphedema Staging System to characterize such affected areas. This study aims to identify risk and protective factors for the development of truncal and upper extremity lymphedema using alternative lymphatic drainage, providing implications for medical and surgical treatment. METHODS: Patients undergoing revisional breast surgery with suspicion of upper extremity lymphedema between 12/2014 and 3/2020 were offered lymphangiography. The breast and lateral/anterior trunks were visualized and blindly evaluated for axillary and inguinal lymphatic flow. A linear-weighted Cohen's kappa statistic was calculated comparing alternative drainage evaluation. Binomial regression was used to compute relative risks (RRs). Significance was assessed at alpha = 0.05. RESULTS: Eighty-six sides (46 patients) were included. Twelve sides underwent no treatment and were considered controls. Eighty-eight percent of the noncontrols had alternative lymphatic flow to the ipsilateral axillae (64%), ipsilateral groins (57%), contralateral axillae (20.3%), and contralateral groins (9.3%). Cohen's kappa for alternative drainage was 0.631 ± 0.043. Ipsilateral axillary and contralateral inguinal drainage were associated with reduced risk of developing truncal lymphedema [RR 0.78, confidence interval (CI) 0.63-0.97, P = 0.04; RR 0.32, CI 0.13-0.79, P = 0.01, respectively]. Radiation therapy increased risk of truncal and upper extremity lymphedema (RR 3.69, CI 0.96-14.15, P = 0.02; RR 1.92, CI 1.09-3.39, P = 0.03, respectively). Contralateral axillary drainage and axillary lymph node dissection were associated with increased risk of upper extremity lymphedema (RR 4.25, CI 1.09-16.61, P = 0.01; RR 2.83, CI 1.23-6.52, P = 0.01, respectively). CONCLUSIONS: Building upon previous work, this study shows risk and protective factors for the development of truncal and upper extremity lymphedema. Most prevalent alternative channels drain to the ipsilateral axilla and groin. Ipsilateral axillary and contralateral inguinal drainage were associated with reduced risk of truncal lymphedema. Patients with radiation, axillary dissection, and contralateral axillary drainage were associated with increased risk of upper extremity lymphedema. These findings have important clinical implications for postoperative manual lymphatic drainage and for determining eligibility for lymphovenous bypass surgery.

Characterization of Atherosclerotic Mice Reveals a Sex-Dependent Susceptibility to Plaque Calcification but No Major Changes in the Lymphatics in the Arterial Wall.

Lymphatics participate in reverse cholesterol transport, and their presence in the arterial wall of the great vessels and prior experimental results suggest their possible role in the development of atherosclerosis. The aim of this study was to characterize the lymphatic vasculature of the arterial wall in atherosclerosis. Tissue sections and tissue-cleared aortas of wild-type mice unveiled significant differences in the density of the arterial lymphatic network throughout the arterial tree. Male and female Ldlr-/- and ApoE-/- mice on a Western diet showed sex-dependent differences in plaque formation and calcification. Female mice on a Western diet developed more calcification of atherosclerotic plaques than males. The lymphatic vessels within the aortic wall of these mice showed no major changes regarding the number of lymphatic junctions and end points or the lymphatic area. However, female mice on a Western diet showed moderate dilation of lymphatic vessels in the abdominal aorta and exhibited indications of increased peripheral lymphatic function, findings that require further studies to understand the role of lymphatics in the arterial wall during the development of atherosclerosis.

Impaired meningeal lymphatic drainage in Listeria monocytogenes infection.

Previous studies have demonstrated an association between lymphatic vessels and diseases caused by bacterial infections. Listeria monocytogenes (LM) bacterial infection can affect multiple organs, including the intestine, brain, liver and spleen, which can be fatal. However, the impacts of LM infection on morphological and functional changes of lymphatic vessels remain unexplored. In this study, we found that LM infection not only induces meningeal and mesenteric lymphangiogenesis in mice, but also impairs meningeal lymphatic vessels (MLVs)-mediated macromolecules drainage. Interestingly, we found that the genes associated with lymphatic vessel development and function, such as Gata2 and Foxc2, were downregulated, suggesting that LM infection may affect cellular polarization and valve development. On the other hand, photodynamic ablation of MLVs exacerbated inflammation and bacterial load in the brain of mice with LM infection. Overall, our findings indicate that LM infection induces lymphangiogenesis and may affect cell polarization, cavity formation, and valve development during lymphangiogenesis, ultimately impairing MLVs drainage.

[Robotic-Assisted Lymphatic Surgery]. / Robotisch-assistierte Lymphchirurgie.

Surgical robotic systems specifically developed for microsurgery are increasingly being used in recent years, particularly in reconstructive lymphatic surgery. Currently, there are two robotic systems that are used in microsurgery. Both systems feature tremor reduction and motion scaling technologies, which are intended to optimise the surgeon's precision and dexterity. In the Department of Plastic Surgery and Hand Surgery at the University Hospital Zurich, the Symani Surgical System is used in many microsurgical and supermicrosurgical procedures. It is mainly used in reconstructive lymphatic surgery, especially for robotic-assisted lymphovenous anastomosis, microvascular anastomosis of lymph node flaps, and it is used in central lymphatic surgery. The robot enables smaller surgical approaches for deep anatomical structures with enhanced surgical precision. In combination with an exoscope, it can also improve the ergonomics of the microsurgeon.

The N-LVA Study: effectiveness and cost-effectiveness of lymphaticovenous anastomosis (LVA) for patients with cancer who suffer from chronic peripheral lymphoedema - study protocol of a multicentre, randomised sham-controlled trial.

INTRODUCTION: Cancer-related lymphoedema is one of the most debilitating side-effects of cancer treatment with an overall incidence of 15.5%. Patients may suffer from a variety of symptoms, possibly resulting in a diminished health-related quality of life (HRQoL). A microsurgical technique known as lymphaticovenous anastomosis (LVA) might be a promising treatment option. The objective of this study is to evaluate whether LVA is effective and cost-effective compared with sham surgery in improving the HRQoL. METHODS AND ANALYSIS: A multicentre, double-blind, randomised sham-controlled trial conducted in three university hospitals in the Netherlands. The study population comprises 110 patients over the age of 18 years with unilateral, peripheral cancer-related lymphoedema, including 70 patients with upper limb lymphoedema and 40 patients with lower limb lymphoedema. A total of 55 patients will undergo the LVA operation, while the remaining 55 will undergo sham surgery. The follow-up will be at least 24 months. Patients are encouraged to complete the follow-up by explaining the importance of the study. Furthermore, patients may benefit from regular monitoring moments for their lymphoedema. The primary outcome is the HRQoL. The secondary outcomes are the limb circumference, excess limb volume, changes in conservative therapy, postoperative complications, patency of the LVA and incremental cost-effectiveness. ETHICS AND DISSEMINATION: The study was approved by the Medical Ethical Committee of Maastricht University Medical Center on 20 September 2023 (NL84169.068.23). The results will be presented at scientific conferences and published in peer-reviewed medical journals. TRIAL REGISTRATION NUMBER: NCT06082349.

Central conducting lymphatic anomaly: from bench to bedside.

Central conducting lymphatic anomaly (CCLA) is a complex lymphatic anomaly characterized by abnormalities of the central lymphatics and may present with nonimmune fetal hydrops, chylothorax, chylous ascites, or lymphedema. CCLA has historically been difficult to diagnose and treat; however, recent advances in imaging, such as dynamic contrast magnetic resonance lymphangiography, and in genomics, such as deep sequencing and utilization of cell-free DNA, have improved diagnosis and refined both genotype and phenotype. Furthermore, in vitro and in vivo models have confirmed genetic causes of CCLA, defined the underlying pathogenesis, and facilitated personalized medicine to improve outcomes. Basic, translational, and clinical science are essential for a bedside-to-bench and back approach for CCLA.

100 anastomoses: a two-year single-center experience with robotic-assisted micro- and supermicrosurgery for lymphatic reconstruction.

Robotic-assisted microsurgery has gained significant attention in recent years following the introduction of two dedicated microsurgical robotic systems specifically designed for this purpose. These feature higher degrees of movement and motion scaling which are useful tools, especially when performing surgery in areas of the body which are difficult to access. Robotic-assisted microsurgery has been implemented in lymphatic surgery as well as soft tissue reconstructive surgery at our institution over the past 2.5 years. Our study gives an insight into the details and outcomes of the first 100 consecutive (super-) microsurgical anastomoses in peripheral and central lymphatic reconstruction performed with the Symani® Surgical System between 2021 and 2024. In total, 67 patients were treated, receiving robotic-assisted lymphatic reconstruction with lymphatic tissue transfer (LTT) and/or lymphovenous anastomoses (LVA)/lympholymphatic anastomoses (LLA). No anastomosis-associated complications were recorded postoperatively. The majority of patients reported a postoperative improvement of their lymphedema or central lymphatic disorder. In conclusion, we show the successful implementation of the Symani® Surgical System into our clinical practice of lymphatic reconstruction. Although the necessary intraoperative setup and the use of intrinsic motion scaling lead to a slight increase in operating time, the presented study demonstrates the advantages of robotic assistance which becomes particularly evident in lymphatic surgery due to the involved deep surgical sites and the need for supermicrosurgical techniques.

Advances in lymphatic metastasis of non-small cell lung cancer.

Lung cancer is a deeply malignant tumor with high incidence and mortality. Despite the rapid development of diagnosis and treatment technology, abundant patients with lung cancer are still inevitably faced with recurrence and metastasis, contributing to death. Lymphatic metastasis is the first step of distant metastasis and an important prognostic indicator of non-small cell lung cancer. Tumor-induced lymphangiogenesis is involved in the construction of the tumor microenvironment, except promoting malignant proliferation and metastasis of tumor cells, it also plays a crucial role in individual response to treatment, especially immunotherapy. Thus, this article reviews the current research status of lymphatic metastasis in non-small cell lung cancer, in order to provide some insights for the basic research and clinical and translational application in this field.

Sensitivity of Each Index of Doppler, Cross, Uncollapsible, Parallel, and Superficial Fascia in Lymphatic Ultrasound.

Background: Recently, the usefulness of lymphatic ultrasound has been reported. It is beneficial not only to identify lymphatic vessels but also to evaluate lymphatic degeneration and diagnose lymphedema. We previously proposed D-CUPS (Doppler, Cross, Uncollapsible, Parallel, and Superficial fascia) to identify the lymphatic vessels on ultrasound. The purpose of this study was to clarify the sensitivity of each index of D-CUPS. Methods: We performed a retrospective study of 27 patients (44 limbs, 98 sites) with lower extremity lymphedema, who underwent lymphaticovenous anastomosis (LVA). We performed a lymphatic ultrasound the day before surgery. We used a linear probe commonly used for venous ultrasound (Noblus EUP-L65; Hitachi Medical Corp., Tokyo, Japan). We applied the D-CUPS index to identify the lymphatic vessels on ultrasound. We checked whether lymphatic vessels consistent with preoperative lymphatic ultrasound findings were observed during the LVA. We also calculated the sensitivity of each D-CUPS index. Results: All the 27 patients were women, with a mean age of 59.7 years. Totally, 98 incisions were made (59 incisions on the thigh and 39 incisions on the lower leg). During LVA, lymphatic vessels consistent with the preoperative lymphatic ultrasound findings were observed at all the sites. The sensitivities of each indicator of D-CUPS were 100.0%, 100.0%, 68.4%, 19.4%, and 100.0%, respectively. Conclusion: The sensitivity was 100.0% in D, C, and S. Although each index separately was not perfect, by combining them appropriately, we were able to identify lymphatic vessels with certainty.

Real-Time Evaluation of Absolute, Cytosolic, Free Ca2+ and Corresponding Contractility in Isolated, Pressurized Lymph Vessels.

The lymphatic vasculature, now often referred to as "the third circulation," is located in many vital organ systems. A principal mechanical function of the lymphatic vasculature is to return fluid from extracellular spaces back to the central venous ducts. Lymph transport is mediated by spontaneous rhythmic contractions of lymph vessels (LVs). LV contractions are largely regulated by the cyclic rise and fall of cytosolic, free calcium ([Ca2+]i). This paper presents a method to concurrently calculate changes in absolute concentrations of [Ca2+]i and vessel contractility/rhythmicity in real time in isolated, pressurized LVs. Using isolated rat mesenteric LVs, we studied changes in [Ca2+]i and contractility/rhythmicity in response to drug addition. Isolated LVs were loaded with the ratiometric Ca2+-sensing indicator Fura-2AM, and video microscopy coupled with edge-detection software was used to capture [Ca2+]i and diameter measurements continuously in real time. The Fura-2AM signal from each LV was calibrated to the minimum and maximum signal for each vessel and used to calculate absolute [Ca2+]i. Diameter measurements were used to calculate contractile parameters (amplitude, end diastolic diameter, end systolic diameter, calculated flow) and rhythmicity (frequency, contraction time, relaxation time) and correlated with absolute [Ca2+]i measurements.