Lymphatic Vessel Function Research Articles

Meningeal lymphatics prime tumor immunity in glioblastoma.

The notion of the brain as an immune-privileged organ has been challenged by the discovery of functional lymphatic vessels in the meninges of the dorsal and basal skull. A study published in Nature in 2020 shows that meningeal lymphatics play an important role in the sensing of brain tumor antigens.

Oxidatively Modified LDL Suppresses Lymphangiogenesis via CD36 Signaling.

Arterial accumulation of plasma-derived LDL and its subsequent oxidation contributes to atherosclerosis. Lymphatic vessel (LV)-mediated removal of arterial cholesterol has been shown to reduce atherosclerotic lesion formation. However, the precise mechanisms that regulate LV density and function in atherosclerotic vessels remain to be identified. The aim of this study was to investigate the role of native LDL (nLDL) and oxidized LDL (oxLDL) in modulating lymphangiogenesis and underlying molecular mechanisms. Western blotting and immunostaining experiments demonstrated increased oxLDL expression in human atherosclerotic arteries. Furthermore, elevated oxLDL levels were detected in the adventitial layer, where LV are primarily present. Treatment of human lymphatic endothelial cells (LEC) with oxLDL inhibited in vitro tube formation, while nLDL stimulated it. Similar results were observed with Matrigel plug assay in vivo. CD36 deletion in mice and its siRNA-mediated knockdown in LEC prevented oxLDL-induced inhibition of lymphangiogenesis. In addition, oxLDL via CD36 receptor suppressed cell cycle, downregulated AKT and eNOS expression, and increased levels of p27 in LEC. Collectively, these results indicate that oxLDL inhibits lymphangiogenesis via CD36-mediated regulation of AKT/eNOS pathway and cell cycle. These findings suggest that therapeutic blockade of LEC CD36 may promote arterial lymphangiogenesis, leading to increased cholesterol removal from the arterial wall and reduced atherosclerosis.

Lymphatic muscle cells contribute to dysfunction of the synovial lymphatic system in inflammatory arthritis in mice

BackgroundOur previous studies reveal that impaired draining function of the synovial lymphatic vessel (LV) contributes to the pathogenesis of inflammatory arthritis, but the cellular and molecular mechanisms involved are not fully understood.ObjectiveTo investigate the involvement of lymphatic muscle cells (LMCs) in mediating impaired LV function in inflammatory arthritis.MethodsTNF transgenic (TNF-Tg) arthritic mice were used. The structure and function of the LVs that drained the hind limbs were examined by whole-mount immunofluorescence staining, electron microscopy, and near-infrared lymphatic imaging. Primary LMCs were treated with TNF, and the changes in proliferation, apoptosis, and functional gene expression were assessed. The roles of the herbal drug, Panax notoginseng saponins (PNS), in arthritis and LVs were studied.ResultsTNF-Tg mice developed ankle arthritis with age, which was associated with abnormalities of LVs: (1) dilated capillary LVs with few branch points, (2) mature LVs with reduced LMC coverage and draining function, and (3) degenerative and apoptotic appearance of LMCs. TNF caused LMC apoptosis, reduced expression of muscle functional genes, and promoted the production of nitric oxide (NO) by lymphatic endothelial cells (LECs). PNS attenuated arthritis, restored LMC coverage and draining function of mature LVs, inhibited TNF-mediated NO expression, and reduced LMC apoptosis.ConclusionThe impaired draining function of LVs in TNF-Tg mice involves LMC apoptosis. TNF promotes LMC death directly and indirectly via NO production by LECs. PNS attenuates arthritis, improves LVs, and prevents TNF-induced LMC apoptosis by inhibiting NO production of LECs. LMCs contribute to the dysfunction of synovial LVs in inflammatory arthritis.

Beyond PROX1: transcriptional, epigenetic, and noncoding RNA regulation of lymphatic identity and function.

The lymphatic vascular system acts as the major transportation highway of tissue fluids, and its activation or impairment is associated with a wide range of diseases. There has been increasing interest in understanding the mechanisms that control lymphatic vessel formation (lymphangiogenesis) and function in development and disease. Here, we discuss recent insights into new players whose identification has contributed to deciphering the lymphatic regulatory code. We reveal how lymphatic endothelial cells, the building blocks of lymphatic vessels, utilize their transcriptional, post-transcriptional, and epigenetic portfolio to commit to and maintain their vascular lineage identity and function, with a particular focus on development.

The evolving cardiac lymphatic vasculature in development, repair and regeneration.

The lymphatic vasculature has an essential role in maintaining normal fluid balance in tissues and modulating the inflammatory response to injury or pathogens. Disruption of normal development or function of lymphatic vessels can have severe consequences. In the heart, reduced lymphatic function can lead to myocardial oedema and persistent inflammation. Macrophages, which are phagocytic cells of the innate immune system, contribute to cardiac development and to fibrotic repair and regeneration of cardiac tissue after myocardial infarction. In this Review, we discuss the cardiac lymphatic vasculature with a focus on developments over the past 5 years arising from the study of mammalian and zebrafish model organisms. In addition, we examine the interplay between the cardiac lymphatics and macrophages during fibrotic repair and regeneration after myocardial infarction. Finally, we discuss the therapeutic potential of targeting the cardiac lymphatic network to regulate immune cell content and alleviate inflammation in patients with ischaemic heart disease.

Multipoint injection indocyanine green lymphography can reduce lymphaticovenular anastomosis surgical time and improve the surgical results for lymphedema

Aim: Lymphaticovenular anastomosis (LVA) is the mainstay of surgical treatment of lymphedema now. Indocyanine green (ICG) lymphography is a method for detecting lymphatic pathways and for the clinical evaluation of patients with extremity lymphedema. The essential point of LVA is to find more functional lymphatic vessels. Sometimes, in cases of lymphatic dysfunction, ICG injections into the distal extremities are insufficient. The purpose of this study was to elucidate the effect of multi-injection of ICG lymphography on LVA.

Prox1-Dependent Stimulation of Lymphatic Function Ameliorates Mesenteric Lesions and Modifies the Composition of Microbiota in Experimental Crohn's Disease

Background: Prox1 is a transcription factor necessary for lymphatic function. The main aim of the current study was to investigate the potential role of Prox1 in lymphangiogenesis and lymphatic function, and analyze Prox1-dependent stimulation of lymphatic function on mesenteric lesions and the composition of microbiota in Crohn’s disease. Methods: Prox1flox/+, Tie2-CreERT2 and IL-10 KO mice were included. Disease activity and enterocolitis inflammation were assessed using a grading system. Lymphatic vessel density and lymphatic function were analyzed using immunohistochemistry and lymphangiography. The potential mechanism of Prox1 was analyzed. The effects of lymphatic function on mesenteric adipose tissue (MAT) and the composition of microbiota were evaluated. Results: Systemic delivery of AAV-Prox1 reduced disease activity index and the severity of chronic inflammation in IL-10 KO mice. Without compensatory response of Prox1, IL-10 KO+Prox1 KO mice developed serious inflammation in the colon. For lymphatic vessel, IL-10 KO+Prox1 KO mice showed lower lymphatic vessel density and less functional lymphatic vessels. IL-10 KO+AAV-Prox1 mice showed significantly increased lymphatic vessel density. Delivery of AAV-Prox1 also promoted lymphatic drainage function. The Prox1/VEGFC/VEGFR3 pathway played an important role in lymphangiogenesis and lymphatic function. Additionally, Prox1-dependent stimulation of lymphatic function ameliorated hypertrophy of MAT in IL-10 KO mice. Delivery of AAV-Prox1 also modified the composition of microbiota, the proportion of Firmicutes increased and Bacteroidetes decreased in IL-10 KO+AAV-Prox1 mice compared with IL-10 KO mice. An increase in the diversity of gut microbiota in IL-10 KO+ AAV-Prox1 mice was observed. Conclusions: Our findings demonstrated that Prox1 played the critical role in the lymphangiogenesis and lymphatic function. Additionally, Prox1-dependent stimulation of lymphatic function could ameliorate hypertrophy of MAT and modify the composition of microbiota in experimental Crohn’s disease. Funding Statement: This work was partly supported by National Natural Science Foundation of China (Grant 81670471, 81770556 and 81570500). Declaration of Interests: The authors declare that they have no conflicts of interests. Ethics Approval Statement: All animal protocols were approved by the Institutional Ethics Committee of the Jinling Hospital, Medical School of Nanjing University, Nanjing, China.

Combined Approach to Surgical Treatment of Lymphedema.

Physiologic surgical interventions, including lymphovenous bypass (LVB) and vascularized lymph node transplant (VLNT), are increasingly being used to treat lymphedema. LVB has been shown to be effective in improving the severity of lymphedema, particularly for patients with still-functional superficial lymphatic vessels that can be identified for bypass. However, in many patients, there is a paucity of functional lymphatic vessels for bypass and, thus, they are not ideal candidates for LVB alone. Unlike LVB, VLNT does not depend on the presence of functioning lymphatic vessels, but the effects of VLNT are delayed, as the proposed mechanisms of action require more time for optimal function. The author has offered a combined approach to microsurgical treatment of lymphedema for both the upper and lower extremities. Simultaneous VLNT and LVB are safe and effective for patients with both early and advanced stages of primary and secondary lymphedema. Our experience shows that a majority of patients can expect some long-term improvement, in both overall limb volume and quality of life, after surgical intervention with LVB and/or VLNT.

Lymphatic and Blood Network Analysis During Obesity.

Lymphatic collecting vessels and lymph nodes are inevitably embedded in adipose tissue. The physiological significance of this observation remains still not elucidated. However, obesity is characterized by impaired lymphatic function and increased vessel permeability. Inversely, lymphatic dysfunction induces obesity in mice, suggesting a significant interplay between lymphatic vessels and the adipose tissue. Therefore, understanding factors leading to lymphatic dysfunction might open new therapeutic windows to prevent obesity and associated comorbidities. The first step in this process requires a precise and detailed visualization of the lymphatic network in healthy and inflamed adipose tissue. Here, we describe a rapid, inexpensive, and efficient method that allows to label and analyze lymphatic and blood vessels. This approach takes advantage of the skin-draining brachial lymph node localization within the subcutaneous adipose tissue. The lymphatic arborization of this tissue can be revealed by injecting fluorochrome-conjugated lectins subcutaneously. Moreover, the in vivo labeling approach provides a way to evaluate lymphatic vessel density and functions. Coupled to blood vessel, adipocyte and immune cell staining, the protocol allows for high-resolution mapping of the subcutaneous adipose tissue by 3D imaging.

Lymphatic drainage from bronchus-associated lymphoid tissue in tolerant lung allografts promotes peripheral tolerance.

Tertiary lymphoid organs are aggregates of immune and stromal cells including high endothelial venules and lymphatic vessels that resemble secondary lymphoid organs and can be induced at nonlymphoid sites during inflammation. The function of lymphatic vessels within tertiary lymphoid organs remains poorly understood. During lung transplant tolerance, Foxp3+ cells accumulate in tertiary lymphoid organs that are induced within the pulmonary grafts and are critical for the local downregulation of alloimmune responses. Here, we showed that tolerant lung allografts could induce and maintain tolerance of heterotopic donor-matched hearts through pathways that were dependent on the continued presence of the transplanted lung. Using lung retransplantation, we showed that Foxp3+ cells egressed from tolerant lung allografts via lymphatics and were recruited into donor-matched heart allografts. Indeed, survival of the heart allografts was dependent on lymphatic drainage from the tolerant lung allograft to the periphery. Thus, our work indicates that cellular trafficking from tertiary lymphoid organs regulates immune responses in the periphery. We propose that these findings have important implications for a variety of disease processes that are associated with the induction of tertiary lymphoid organs.

Magnetic resonance lymphography as three-dimensional navigation for lymphaticovenular anastomosis in patients with leg lymphedema

Precise mapping of functional lymphatic vessels is essential for successful lymphaticovenular anastomosis (LVA). This study aimed to clarify the precision of magnetic resonance lymphography (MRL) in detecting lymphatic vessels prior to LVA. Eighteen patients with leg lymphedema were recruited for this prospective study. All patients underwent MRL before LVA to obtain three-dimensional coordinates of lymphatic vessels from MRL images. The precision of MRL for detecting lymphatic vessels was evaluated and compared with those of other contrast techniques. Twenty legs from 18 patients were analyzed. A total of 40 skin incisions were made, 32 of which were determined by MRL. The precision of MRL to detect lymphatic vessels was 94%. With the addition of MRL, the number of lymphatic vessels identified preoperatively was increased as compared with indocyanine green lymphography (ICG-L) alone. Assuming a detection sensitivity of MRL for lymphatic vessels of 1, those of other contrast techniques were 0.90 for ICG-L under microscopy, 0.73 for patent blue staining, and 0.43 for ICG-L before incision. Whereas ICG-L before incision could not detect lymphatic vessels at depths greater than 17.0 mm, all deeper anastomosed lymphatic vessels were identified by MRL. Lymphatic vessels enhanced on MRL can be reliably identified intraoperatively. MRL is a promising preoperative examination in LVA that can selectively depict suitable lymphatic vessels even in deep tissue layers.

Renal lymphatic vessel dynamics.

Similar to other organs, renal lymphatics remove excess fluid, solutes, and macromolecules from the renal interstitium. Given the kidney's unique role in maintaining body fluid homeostasis, renal lymphatics may be critical in this process. However, little is known regarding the pathways involved in renal lymphatic vessel function, and there are no studies on the effects of drugs targeting impaired interstitial clearance, such as diuretics. Using pressure myography, we showed that renal lymphatic collecting vessels are sensitive to changes in transmural pressure and have an optimal range of effective pumping. In addition, they are responsive to vasoactive factors known to regulate tone in other lymphatic vessels including prostaglandin E2 and nitric oxide, and their spontaneous contractility requires Ca2+ and Cl-. We also demonstrated that Na+-K+-2Cl- cotransporter Nkcc1, but not Nkcc2, is expressed in extrarenal lymphatic vessels. Furosemide, a loop diuretic that inhibits Na+-K+-2Cl- cotransporters, induced a dose-dependent dilation in lymphatic vessels and decreased the magnitude and frequency of spontaneous contractions, thereby reducing the ability of these vessels to propel lymph. Ethacrynic acid, another loop diuretic, had no effect on vessel tone. These data represent a significant step forward in our understanding of the mechanisms underlying renal lymphatic vessel function and highlight potential off-target effects of furosemide that may exacerbate fluid accumulation in edema-forming conditions.

Structure and physiology of the lymphatic vasculature

The lymphatic vascular system is a highly organized network of structurally and functionally connected specialized lymphatic vessels of various sizes and lymph nodes that perform metabolic and transport functions. Lymph is a blood plasma filtrate that comprises antigen-presenting cells and lymphocytes. Via lymph, excess fluid and extravasated proteins are removed from the tissues. The lymphatic system supports an extracellular fluid homeostasis that is favorable for optimal tissue functioning by removing substances that result from metabolism or cell death, as well as optimizing immunity against bacteria, viruses and other antigens. Although the lymphatic vasculature is not formally considered part of the immune system, it is crucial for the traffic of antigens and immune cells. In addition, lymphatic endothelial cells can supply antigens and express factors that modulate immune responses. After an inflammatory stimulus, endothelial cells produce chemokines, which recruit immune cells to the lymph nodes. Unlike the circulatory system with a centralized pump, the movement of lymph through the network of lymphatic vessels is provided by forces that stimulate the initial formation of lymph in the tissues and the ability of the lymphatic vessels and nodes to rhythmically contract, providing increased pressure and lymph movement in the proximal direction. Since the metabolic rate in various organs and tissues varies significantly depending on the functional state of the tissue, the blood flow through the tissue and the amount of lymph formed also change significantly. The lymphatic vasculature has several circuits for regulating lymph flow. This review provides a comprehensive overview of the important results obtained over the past century and discusses the molecular and physiological control of the transport function of lymphatic vessels and nodes.

Changing the Paradigm: Lymphovenous Anastomosis in Advanced Stage Lower Extremity Lymphedema.

Traditionally, lymphovenous anastomosis is not routinely performed in patients with advanced stage lymphedema because of difficulty with identifying functioning lymphatics. This study presents the use of duplex ultrasound and magnetic resonance lymphangiography to identify functional lymphatics and reports the clinical outcome of lymphovenous anastomosis in advanced stage lower extremity lymphedema patients. This was a retrospective study of 42 patients (50 lower limbs) with advanced lymphedema (late stage 2 or 3) that underwent functional lymphovenous anastomoses. Functional lymphatic vessels were identified preoperatively using magnetic resonance lymphangiography and duplex ultrasound. An average of 4.64 lymphovenous anastomoses were performed per limb using the lymphatics located in the deep fat underneath the superficial fascia. The average diameter of lymphatic vessels was 0.61 mm (range, 0.35 to 1 mm). The average limb volume was reduced 14.0 percent postoperatively, followed by 15.2 percent after 3 months, and 15.5 percent after 6 months and 1 year (p < 0.001). For patients with unilateral lymphedema, 32.4 percent had less than 10 percent volume excess compared to the contralateral side postoperatively, whereas 20.5 percent had more than 20 percent volume excess. The incidence of cellulitis decreased from 0.84 per year to 0.07 per year after surgery (p < 0.001). This study shows that functioning lymphatic vessels can be identified preoperatively using ultrasound and magnetic resonance lymphangiography; thus, lymphovenous anastomoses can effectively reduce the volume of the limb and improve subjective symptoms in patients with advanced stage lymphedema of the lower extremity. Therapeutic, IV.

Chylomicrons-Simulating Sustained Drug Release in Mesenteric Lymphatics for the Treatment of Crohn's-Like Colitis.

Alteration to both the structures and functions of mesenteric lymphatic vessels is a typical hallmark of Crohn's disease [CD]. Dysfunctional lymphatics was observed in patients with both CD and experimental colitis, suggesting mesenteric lymphatics could be potential therapeutic targets. This study aimed to develop a nano-delivery system which can enhance drug delivery in mesenteric lymphatic tissue [MLT] and evaluate the therapeutic effects in Crohn's colitis. We designed a mesoporous silica nanoparticle [MSN] conjugated with long-chain fatty acid [LMSN] and covered with enteric coating [ELMSN] which can be specifically transported via the mesenteric lymphatic system. The therapeutic efficacy of laquinimod-loaded nanoparticles [LAQ@ELMSN] was evaluated in the well-established interleukin [IL]-10-/- spontaneous experimental colitis. ELMSNs induced sustainable drug release that markedly increased drug concentration in MLT. In experimental colitis, the lymphatics-targeting drug delivery system suppressed lymphangitis and promoted lymphatic drainage. The downregulation of pro-inflammatory cytokines and the downstream NF-κB-related proteins efficiently inhibited lymphangiogenesis and restored tight junctions of mesenteric lymphatic vessels [MLVs]. LAQ@ELMSN showed a superior therapeutic effect in ameliorating intestinal inflammation compared with free drug administration. Alteration of gut microbiota and metabolites in experimental colitis was also reversed by LAQ@ELMSN. Our study demonstrates a convenient, orally administered drug delivery system which enhances drug release in MLT. The results confirm the contribution of the mesenteric lymphatic system to the pathogenesis of gut inflammation and shed light on the application of lymphatics-targeting drug delivery therapy as a potential therapeutic strategy for CD treatment.

EphrinB2-EphB4 signalling provides Rho-mediated homeostatic control of lymphatic endothelial cell junction integrity.

Endothelial integrity is vital for homeostasis and adjusted to tissue demands. Although fluid uptake by lymphatic capillaries is a critical attribute of the lymphatic vasculature, the barrier function of collecting lymphatic vessels is also important by ensuring efficient fluid drainage as well as lymph node delivery of antigens and immune cells. Here, we identified the transmembrane ligand EphrinB2 and its receptor EphB4 as critical homeostatic regulators of collecting lymphatic vessel integrity. Conditional gene deletion in mice revealed that EphrinB2/EphB4 signalling is dispensable for blood endothelial barrier function, but required for stabilization of lymphatic endothelial cell (LEC) junctions in different organs of juvenile and adult mice. Studies in primary human LECs further showed that basal EphrinB2/EphB4 signalling controls junctional localisation of the tight junction protein CLDN5 and junction stability via Rac1/Rho-mediated regulation of cytoskeletal contractility. EphrinB2/EphB4 signalling therefore provides a potential therapeutic target to selectively modulate lymphatic vessel permeability and function.

Biological functions of lymphatic vessels.

The general functions of lymphatic vessels in fluid transport and immunosurveillance are well recognized. However, accumulating evidence indicates that lymphatic vessels play active and versatile roles in a tissue- and organ-specific manner during homeostasis and in multiple disease processes. This Review discusses recent advances to understand previously unidentified functions of adult mammalian lymphatic vessels, including immunosurveillance and immunomodulation upon pathogen invasion, transport of dietary fat, drainage of cerebrospinal fluid and aqueous humor, possible contributions toward neurodegenerative and neuroinflammatory diseases, and response to anticancer therapies.

Extracellular Vesicles as Potential Prognostic Markers of Lymphatic Dysfunction.

Despite significant efforts made to treat cardiovascular disease (CVD), more than half of cardiovascular events still occur in asymptomatic subjects devoid of traditional risk factors. These observations underscore the need for the identification of new biomarkers for the prevention of atherosclerosis, the main underlying cause of CVD. Extracellular vesicles (EVs) and lymphatic vessel function are emerging targets in this context. EVs are small vesicles released by cells upon activation or death that are present in several biological tissues and fluids, including blood and lymph. They interact with surrounding cells to transfer their cargo, and the complexity of their biological content makes these EVs potential key players in several chronic inflammatory settings. Many studies focused on the interaction of EVs with the most well-known players of atherosclerosis such as the vascular endothelium, smooth muscle cells and monocytes. However, the fate of EVs within the lymphatic network, a crucial route in the mobilization of cholesterol out the artery wall, is not known. In this review, we aim to bring forward evidence that EVs could be at the interplay between lymphatic function and atherosclerosis by summarizing the recent findings on the characterization of EVs in this setting.

Blocking Lymph Flow by Suturing Afferent Lymphatic Vessels in Mice.

Lymphatic vessels are critical in maintaining tissue fluid balance and optimizing immune protection by transporting antigens, cytokines, and cells to draining lymph nodes (LNs). Interruption of lymph flow is an important method when studying the function of lymphatic vessels. The afferent lymphatic vessels from the murine footpad to the popliteal lymph nodes (pLNs) are well-defined as the only routes for lymph drainage into the pLNs. Suturing these afferent lymphatic vessels can selectively prevent lymph flow to the pLNs. This method allows for interference in lymph flow with minimal damage to the lymphatic endothelial cells in the draining pLN, the afferent lymphatic vessels, as well as other lymphatic vessels around the area. This method has been used to study how lymph impacts high endothelial venules (HEV) and chemokine expression in the LN, and how lymph flows through the adipose tissue surrounding the LN in the absence of functional lymphatic vessels. With the growing recognition of the importance of lymphatic function, this method will have broader applications to further unravel the function of lymphatic vessels in regulating the LN microenvironment and immune responses.

Beyond a Passive Conduit: Implications of Lymphatic Biology for Kidney Diseases.

The kidney contains a network of lymphatic vessels that clear fluid, small molecules, and cells from the renal interstitium. Through modulating immune responses and via crosstalk with surrounding renal cells, lymphatic vessels have been implicated in the progression and maintenance of kidney disease. In this Review, we provide an overview of the development, structure, and function of lymphatic vessels in the healthy adult kidney. We then highlight the contributions of lymphatic vessels to multiple forms of renal pathology, emphasizing CKD, transplant rejection, and polycystic kidney disease and discuss strategies to target renal lymphatics using genetic and pharmacologic approaches. Overall, we argue the case for lymphatics playing a fundamental role in renal physiology and pathology and treatments modulating these vessels having therapeutic potential across the spectrum of kidney disease.