Lymphatic Physiology Research Articles

MR Lymphangiography in Lymphatic Disorders: Clinical Applications, Institutional Experience, and Practice Development.

Lymphatic flow and anatomy can be challenging to study, owing to variable lymphatic anatomy in patients with diverse primary or secondary lymphatic pathologic conditions and the fact that lymphatic imaging is rarely performed in healthy individuals. The primary components of the lymphatic system outside the head and neck are the peripheral, retroperitoneal, mesenteric, hepatic, and pulmonary lymphatic systems and the thoracic duct. Multiple techniques have been developed for imaging components of the lymphatic system over the past century, with trade-offs in spatial, temporal, and contrast resolution; invasiveness; exposure to ionizing radiation; and the ability to obtain information on dynamic lymphatic flow. More recently, dynamic contrast-enhanced (DCE) MR lymphangiography (MRL) has emerged as a valuable tool for imaging both lymphatic flow and anatomy in a variety of congenital and acquired primary or secondary lymphatic disorders. The authors provide a brief overview of lymphatic physiology, anatomy, and imaging techniques. Next, an overview of DCE MRL and the development of an MRL practice and workflow in a hybrid interventional MRI suite incorporating cart-based in-room US is provided, with an emphasis on multidisciplinary collaboration. The spectrum of congenital and acquired lymphatic disorders encountered early in an MRL practice is provided, with emphasis on the diversity of imaging findings and how DCE MRL can aid in diagnosis and treatment of these patients. Methods such as DCE MRL for assessing the hepatic and mesenteric lymphatic systems and emerging technologies that may further expand DCE MRL use such as three-dimensional printing are introduced. ©RSNA, 2024 Test Your Knowledge questions for this article are available in the supplemental material.

Murine Dermal Lymphatic Endothelial Cell Isolation.

The lymphatic system participates in the regulation of immune surveillance, lipid absorption, and tissue fluid balance. The isolation of murine lymphatic endothelial cells is an important process for lymphatic research, as it allows the performance of in vitro and biochemical experiments on the isolated cells. Moreover, the development of Cre-lox technology has enabled the tissue-specific deficiency of genes that cannot be globally targeted, leading to the precise determination of their role in the studied tissues. The dissection of the role of certain genes in lymphatic physiology and pathophysiology requires the use of lymphatic-specific promoters, and thus, the experimental verification of the expression levels of the targeted genes. Methods for efficient isolation of lymphatic endothelial cells from wild-type or transgenic mice enable the use of ex vivo and in vitro assays to study the mechanisms regulating the lymphatic functions and the identification of the expression levels of the studied proteins. We have developed, standardized and present a protocol for the efficient isolation of murine dermal lymphatic endothelial cells (DLECs) via magnetic bead purification based on LYVE-1 expression. The protocol outlined aims to equip researchers with a tool to further understand and elucidate important players of lymphatic endothelial cell functions, especially in facilities where fluorescence-activated cell sorting equipment is not available.

Microanatomical organization of hepatic venous lymphatic system in humans.

Lymphatic fluid drains from the liver via the periportal lymphatic, hepatic venous lymphatic, and superficial lymphatic systems. We performed a postmortem study to clarify the three-dimensional structure and flow dynamics of the human hepatic venous lymphatic system, as it still remains unclear. Livers were excised whole from three human cadavers, injected with India ink, and sliced into 1-cm sections from which veins were harvested. The distribution of lymphatic vessels was observed in 5 μm sections immunostained for lymphatic and vascular markers (podoplanin and CD31, respectively) using light microscopy. Continuity and density of lymphatic vessel distribution were assessed in en-face whole-mount preparations of veins using stereomicroscopy. The structure of the external hepatic vein wall was assessed with scanning electron microscopy (SEM). The lymphatic dynamics study suggested that lymphatic fluid flows through an extravascular pathway around the central and sublobular veins. A lymphatic vessel network originates in the wall of sublobular veins, with a diameter greater than 110 μm, and the peripheral portions of hepatic veins and continues to the inferior vena cava. The density distribution of lymphatic vessels is smallest in the peripheral portion of the hepatic vein (0.03%) and increases to the proximal portion (0.22%, p = 0.012) and the main trunk (1.01%, p < 0.001), correlating positively with increasing hepatic vein diameter (Rs = 0.67, p < 0.001). We revealed the three-dimensional structure of the human hepatic venous lymphatic system. The results could improve the understanding of lymphatic physiology and liver pathology.

Human collecting lymphatic glycocalyx identification by electron microscopy and immunohistochemistry

Blood flow is translated into biochemical inflammatory or anti-inflammatory signals based onshear stress type, by means of sensitive endothelial receptors. Recognition of the phenomenon is of paramount importance for enhanced insights into the pathophysiological processes of vascular remodeling. The endothelial glycocalyx is a pericellular matrix, identified in both arteries and veins, acting collectively as a sensor responsive to blood flow changes. Venous and lymphatic physiology is interconnected; however, to our knowledge, a lymphatic glycocalyx structure has never been identified in humans. The objective of this investigation is to identify glycocalyx structures from ex vivo lymphatic human samples. Lower limb vein and lymphatic vessels were harvested. The samples were analyzed by transmission electron microscopy. The specimens were also examined by immunohistochemistry. Transmission electron microscopy identified a glycocalyx structure in human venous and lymphatic samples. Immunohistochemistry for podoplanin, glypican-1, mucin-2, agrin and brevican characterized lymphatic and venous glycocalyx-like structures. To our knowledge, the present work reports the first identification of a glycocalyx-like structure in human lymphatic tissue. The vasculoprotective action of the glycocalyx could become an investigational target in the lymphatic system as well, with clinical implications for the many patients affected by lymphatic disorders.

Locoregional Lymphatic Delivery Systems Using Nanoparticles and Hydrogels for Anticancer Immunotherapy.

The lymphatic system has gained significant interest as a target tissue to control cancer progress, which highlights its central role in adaptive immune response. Numerous mechanistic studies have revealed the benefits of nano-sized materials in the transport of various cargos to lymph nodes, overcoming barriers associated with lymphatic physiology. The potential of sustained drug delivery systems in improving the therapeutic index of various immune modulating agents is also being actively discussed. Herein, we aim to discuss design rationales and principles of locoregional lymphatic drug delivery systems for invigorating adaptive immune response for efficient antitumor immunotherapy and provide examples of various advanced nanoparticle- and hydrogel-based formulations.

Draining the Pleural Space: Lymphatic Vessels Facing the Most Challenging Task.

Simple SummaryFluid drainage operated by lymphatic vessels is crucial for a proper volume homeostasis of body compartments. This role is particularly relevant for the pleural cavity, where the hydraulic pressure of the pleural liquid is very subatmospheric and fluid filtering from the blood capillaries into the pleural space must be continuously removed to keep the pleural space volume low and to prevent accumulation of liquid causing impairments of the respiratory mechanics. In order to accomplish this task, lymphatic vessels of the pleural side of the diaphragm and those lying on the pleural surface of the chest wall must possess a negative intraluminal pressure which has to vary during the respiratory cycle to follow the similar variations occurring to the pressure of pleural liquid. This review focuses on the in vivo pressure measurements performed in sedated animal models to understand how these lymphatic networks can accomplish this complex but pivotal role.Lymphatic vessels exploit the mechanical stresses of their surroundings together with intrinsic rhythmic contractions to drain lymph from interstitial spaces and serosal cavities to eventually empty into the blood venous stream. This task is more difficult when the liquid to be drained has a very subatmospheric pressure, as it occurs in the pleural cavity. This peculiar space must maintain a very low fluid volume at negative hydraulic pressure in order to guarantee a proper mechanical coupling between the chest wall and lungs. To better understand the potential for liquid drainage, the key parameter to be considered is the difference in hydraulic pressure between the pleural space and the lymphatic lumen. In this review we collected old and new findings from in vivo direct measurements of hydraulic pressures in anaesthetized animals with the aim to better frame the complex physiology of diaphragmatic and intercostal lymphatics which drain liquid from the pleural cavity.

All Edema Is Lymphedema: Progressing Lymphedema and Wound Management to an Integrated Model of Care

BACKGROUND: Chronic edema affects millions of people in the United States and worldwide. Edema can result from a variety of diseases, trauma, medications, and other contributing factors; however, all edema is related to lymphatic fluid dysregulation. Additionally, lymphatic impairment and integumentary dysfunction are interrelated, leading to complex clinical presentations that require an integrated medical model of care to maximize outcomes. PURPOSE: This narrative review article will highlight the current evidence that details lymphatic physiology, fluid regulation by the endothelial glycocalyx layer, and the interconnectedness of the vascular and integumentary systems leading to a paradigm shift in our understanding of edema, lymphedema, and chronic wounds. Traditional pedagogy remains siloed with respect to the body systems, whereas current evidence indicates a certain interdependence, particularly between and among the venous, lymphatic, and integumentary systems. METHODS: Comprehensive narrative review of the current and past literature (2010–2021 through PubMed, Google Scholar, MEDLINE Complete, UpToDate) focusing on lymphatic physiology, fluid regulation, the endothelial glycocalyx layer, lymphedema, and venous insufficiency. Review focuses on new evidence supporting the interconnectedness of the systems to support a unified medical management approach. RESULTS: All edema is related to lymphatic dysfunction, whether transient or permanent, thereby creating a lymphedema continuum. Further, lymphatic impairment creates cutaneous regions of skin barrier failure, rendering the skin more susceptible to breakdown and chronic wounds. CONCLUSION: A synthesis of the current evidence suggests an interconnected relationship of the lymphatic, venous, and integumentary systems, highlighting the need for a more integrated medical model of care to provide efficient and comprehensive care and improve patient outcomes.

Post-traumatic lymphedema: review of the literature and surgical treatment options

Post-traumatic lymphedema (PTL) is a complex, debilitating, and potentially common disease which has received limited attention to date. The available literature is reviewed to identify injury patterns and critical lymphatic areas associated with the disease. A deeper understanding of these critical anatomic regions allows the reconstructive surgeon to potentially identify PTL patients earlier in order to apply surgical and nonsurgical interventions in the acute phase, improving lymphatic physiology and, ultimately, patient outcomes. Current diagnostic and treatment approaches are discussed in detail, with a focus on lymphatic microsurgical techniques developed and applied to PTL within the last decade.

A bioengineered lymphatic vessel model for studying lymphatic endothelial cell-cell junction and barrier function.

Lymphatic vessels (LVs) maintain fluid homeostasis by draining interstitial fluid. A failure in lymphatic drainage triggers lymphatic diseases such as lymphedema. Since lymphatic drainage is regulated by lymphatic barrier function, developing experimental models that assess lymphatic barrier function is critical for better understanding of lymphatic physiology and disease. We built a lymphatic vessel-on-chip (LV-on-chip) by fabricating a microfluidic device that includes a hollow microchannel embedded in three-dimensional (3D) hydrogel. Employing luminal flow in the microchannel, human lymphatic endothelial cells (LECs) seeded in the microchannel formed an engineered LV exhibiting 3D conduit structure. Lymphatic endothelial cells formed relatively permeable junctions in 3D collagen 1. However, adding fibronectin to the collagen 1 apparently tightened LEC junctions. We tested lymphatic barrier function by introducing dextran into LV lumens. While LECs in collagen 1showed permeable barriers, LECs in fibronectin/collagen 1showed reduced permeability, which was reversed by integrin α5 inhibition. Mechanistically, LECs expressed inactivated integrin α5 in collagen 1. However, integrin α5 is activated in fibronectin and enhances barrier function. Integrin α5 activation itself also tightened LEC junctions in the absence of fibronectin. Lymphatic vessel-on-chip reveals integrin α5 as a regulator of lymphatic barrier function and provides a platform for studying lymphatic barrier function in various conditions.

Lymphatic impairment leads to pulmonary tertiary lymphoid organ formation and alveolar damage.

The lung is a specialized barrier organ that must tightly regulate interstitial fluid clearance and prevent infection in order to maintain effective gas exchange. Lymphatic vessels are important for these functions in other organs, but their roles in the lung have not been fully defined. In the present study, we addressed how the lymphatic vasculature participates in lung homeostasis. Studies using mice carrying a lymphatic reporter allele revealeded that, in contrast to other organs, lung lymphatic collecting vessels lack smooth muscle cells entirely, suggesting that forward lymph flow is highly dependent on movement and changes in pressure associated with respiration. Functional studies using CLEC2-deficient mice in which lymph flow is impaired due to loss of lympho-venous hemostasis or using inducible lung-specific ablation of lymphatic endothelial cells in a lung transplant model revealeded that loss of lymphatic function leads to an inflammatory state characterized by the formation of tertiary lymphoid organs (TLOs). In addition, impaired lymphatic flow in mice resulteds in hypoxia and features of lung injury that resemble emphysema. These findings reveal both a lung-specific mechanism of lymphatic physiology and a lung-specific consequence of lymphatic dysfunction that may contribute to chronic lung diseases that arise in association with TLO formation.

The Intestinal Lymphatic System: Functions and Metabolic Implications.

The lymphatic system of the gut plays important roles in the transport of dietary lipids, as well as in immunosurveillance and removal of interstitial fluid. Historically, despite its crucial functions in intestinal homeostasis, the lymphatic system has been poorly studied. In the last 2 decades, identification of specific molecular mediators of lymphatic endothelial cells (LECs) growth together with novel genetic approaches and intravital imaging techniques, have advanced our understanding of the mechanisms regulating intestinal lymphatic physiology in health and disease. As its metabolic implications are gaining recognition, intestinal lymphatic biology is currently experiencing a surge in interest. This review describes current knowledge related to molecular control of intestinal lymphatic vessel structure and function. We discuss regulation of chylomicron entry into lymphatic vessels by vascular endothelial growth factors (VEGFs), hormones, transcription factors and the specific signaling pathways involved. The information covered supports the emerging role of intestinal lymphatics in etiology of the metabolic syndrome and their potential as a therapeutic target.

Dietary restriction and aerobic exercise attenuate obesity‐induced lymphatic dysfunction

Abstract Obesity is a deleterious condition characterized by excess adipose tissue deposition and a body mass index greater than 30.0. It is a major risk factor for many diseases, including coronary heart disease and non-insulin dependent diabetes mellitus. Obesity leads to systemic low-grade inflammation caused by cytokines and adipokines. Although obesity has effects on several bodily systems, the lymphatic system is especially important since its relationship with obesity is bidirectional. Obesity is the major contributing factor to the progression and development of lymphedema and impaired lymphatic function is thought to regulate the pathology of obesity in organ systems via modulation of the differentiation and migration of inflammatory cells. It has been shown obesity leads to lymphatic dysfunction which is characterized by impaired lymphatic vessel pumping and density, increased lymphatic vessel leakiness, and altered lymphatic endothelial cell gene expression (Savetsky et al. 2014). Despite the clinical importance of lymphatic physiology in obese patients, an effective therapeutic strategy to regulate lymphatic function remains to be determined. This article is protected by copyright. All rights reserved.

Understanding the Concepts and Physiologic Principles of Lymphatic Microsurgery

Surgical techniques such as lymphaticovenous anastomosis and vascularized lymph node (VLN) transfer are gaining momentum in the treatment of lymphedema. Although surgeons may be technically capable of performing these complex procedures, understanding the normal physiology of the lymphatic system and the pathophysiology of lymphedema is critical to the patient selection process, determining which specific procedure to perform, considering the specific anatomic location of surgery, and optimization of outcomes. To familiarize the plastic surgeon with the pathophysiological principles of lymphatic surgery for lymphedema, this review focuses on lymphatic anatomy, histology, physiology, disease progression, and staging in the context of lymphaticovenous anastomosis and VLN transfer.

Abstract 18661: Regulation of Lymphatic Physiology by Connexin47

Introduction: Mutations in GJC2, which encodes connexin47 (Cx47), are associated with lymphedema. Moreover, Cx47 mRNA was recently found in human lymphatic endothelial cells (LECs). Hypothesis: Lymphatic endothelial Cx47 has a role in lymphatic physiology and pathology. Methods: Confocal microscopy and qPCRs were used to assess expression of Cx47. Lymphatic drainage was studied by subcutaneous Evans Blue injections in the footpad of 12 week-old ApoE -/- or Cx47 eGFP/eGFP ApoE -/- mice. Atherosclerosis was studied in 15 months-old ApoE -/- or Cx47 eGFP/eGFP ApoE -/- mice. Results: Cx47 was expressed in lymphangions of mesenteric collecting vessels in Cx47 eGFP/eGFP Prox1-mOrange2 mice as assessed by microscopy and its expression was confirmed by qPCR on mesentery, gut and skin mRNA of ApoE -/- but not Cx47 eGFP/eGFP ApoE -/- mice. Interestingly, lymphatic drainage was enhanced in Cx47-deficient mice (n=10/11, p&lt;0.05). To address whether Cx47 affects chronic immuno-inflammatory pathology atherosclerotic plaque formation was studied in 15-month old male ApoE -/- and Cx47 eGFP/eGFP ApoE -/- mice on regular chow. These studies showed that Cx47-deficiency does not affect the size or composition (e.g. lipid, collagen or macrophage content) of aortic plaques in the aortic root. However, plaque development in the thoracic-abdominal aorta of 15 months-old mice tended to be larger in Cx47 eGFP/eGFP ApoE -/- mice (21±5 vs 30±4 % lipid area, n=10, p=0.08). Unexpectedly, total cholesterol (TC) and LDL, but not HDL, triglycerides or free fatty acids, were increased in serum of Cx47 eGFP/eGFP ApoE -/- mice as compared to ApoE -/- serum (TC: 472±24 vs 647±18 mg/dL, n=10, p&lt;0.01; and LDL: 272±20 vs 443±17 mg/dL, n=10, p&lt;0.001, respectively). Increased lymphatic drainage from plaques in Cx47 eGFP/eGFP ApoE -/- mice may, partially, compensate for increased serum TC and LDL thereby preventing exuberant atherosclerotic plaque formation despite increased serum lipid levels. Conclusions: In conclusion, Cx47 is expressed in LECs of the lymphangion and seems implicated in multiple aspects of lymphatic (patho)physiology.

Proposed new lymphology combined with lymphatic physiology, innate immunology, and oncology.

As one of the lymphatic functions, it is well known that the transport and drainage of hydrophilic substances including plasma protein through the lymphatic system play pivotal roles in maintaining the homeostasis of the internal environment between the cells in tissues in collaboration with the exchange of the substances through the blood capillaries and venules. The physiological functions of the lymphatic system have been studied by many investigations of microcirculation, i.e., Yoffey and Courtice, Ruszunyak et al., Földie and Casley-Smigh et al., Roddie, Schmid-Schönbein et al., and Ohhashi et al. On the other hand, it is also well known that the initial clinical signs of primary diseases such as inflammation, tumors, and circulatory disorders including infarction and thrombosis appear as functional abnormalities of the internal environment in tissues. These abnormalities of the functions are strongly related to immunological defense reactions around the internal environment and abnormal actions of the transport and drainage of the lymphatic system. Taking into consideration the current inspired findings in lymphatic physiology, innate immunology, and oncology, we have proposed a new lymphology combined with new knowledge of the three above-mentioned academic fields from a defense mechanism points of view. In this review, we would like to demonstrate comprehensively our latest studies related to the possibility of establishing a new lymphology, hoping the readers will evaluate this possibility.

Connexins in lymphatic vessel physiology and disease

Connexins are transmembrane proteins that form gap junction- and hemi-channels. Once inserted into the membrane, hemi-channels (connexons) allow for diffusion of ions and small molecules (<1 kDa) between the extracellular space and the cytosol. Gap junction channels allow diffusion of similar molecules between the cytoplasms of adjacent cells. The expression and function of connexins in blood vessels has been intensely studied in the last few decades. In contrast, only a few studies paid attention to lymphatic vessels; convincing in vivo data with respect to expression patterns of lymphatic connexins and their functional roles have only recently begun to emerge. Interestingly, mutations in connexin genes have been linked to diseases of lymphatic vasculature, most notably primary and secondary lymphedema. This review summarizes the available data regarding lymphatic connexins. More specifically it addresses (i) early studies aimed at presence of gap junction-like structures in lymphatic vessels, (ii) more recent studies focusing on lymphatic connexins using genetically engineered mice, and (iii) results of clinical studies that have reported lymphedema-linked mutations in connexin genes.

Minimally invasive method for determining the effective lymphatic pumping pressure in rats using near-infrared imaging

The ability to quantify collecting vessel function in a minimally invasive fashion is crucial to the study of lymphatic physiology and the role of lymphatic pump function in disease progression. Therefore, we developed a highly sensitive, minimally invasive research platform for quantifying the pumping capacity of collecting lymphatic vessels in the rodent tail and forelimb. To achieve this, we have integrated a near-infrared lymphatic imaging system with a feedback-controlled pressure cuff to modulate lymph flow. After occluding lymphatic flow by inflating a pressure cuff on the limb or tail, we gradually deflate the cuff while imaging flow restoration proximal to the cuff. Using prescribed pressure applications and automated image processing of fluorescence intensity levels in the vessels, we were able to noninvasively quantify the effective pumping pressure (P(eff), pressure at which flow is restored after occlusion) and vessel emptying rate (rate of fluorescence clearance during flow occlusion) of lymphatics in the rat. To demonstrate the sensitivity of this system to changes in lymphatic function, a nitric oxide (NO) donor cream, glyceryl trinitrate ointment (GTNO), was applied to the tails. GTNO decreased P(eff) of the vessels by nearly 50% and the average emptying rate by more than 60%. We also demonstrate the suitability of this approach for acquiring measurements on the rat forelimb. Thus, this novel research platform provides the first minimally invasive measurements of P(eff) and emptying rate in rodents. This experimental platform holds strong potential for future in vivo studies that seek to evaluate changes in lymphatic health and disease.

New Lymphology Combined with Lymphatic Physiology, Innate Immunology, and Oncology

In more than 85 year-old history of the Gordon Research Conference, a conference on the scheme of lymphatic systemwas held for the first time on March 2004, in Ventura, California, USA. Such trends of lymphatic Gordon conference are considered to reflect rapid progress of lymphology inspired by the discovery of new markers of lymphatic endothelial cells such as LYVE-1, podoplanin, and Prox1, and then the development of the VEGF C/D and VEGF R3-mediated lymphangiogenesis and lymphatic metastasis of carcinoma cells.

Basal nitric oxide production in mouse collecting lymphatics does not enhance contractile activity

The mechanisms governing spontaneous contractions in lymphatic vessels are incompletely understood. Accruing evidence in vivo suggests that basal levels of nitric oxide (NO) enhance lymphatic contraction amplitude, despite its well‐defined actions to inhibit contractile activity at moderate doses. Testing this hypothesis, we developed a method to isolate and cannulate collecting lymphatic vessels from the popliteal fossae of wild‐type (WT, n=12) and eNOS−/− mice (n=10). Diastolic diameter, contraction amplitude, and frequency were measured as a function of known pressure (1–10 cmH2O) in the absence of flow, first without then with the NOS inhibitor, L‐NAME. Vessels exhibited robust spontaneous contractions (53% of resting diameter) modulated by pressure, similar to other mammalian species. Contrary to current thought, inhibition of eNOS with L‐NAME (100μM) significantly increased the ejection fraction of WT vessels at low filling pressures (by 28% at 1–3 cmH2O, P&lt;0.05); likewise genetic deletion of eNOS significantly (P&lt;0.05) increased the contraction amplitude (by 45%) and ejection fraction (by 33%). Thus, when pressure and flow are controlled, basal NO production solely depresses lymphatic contractile activity. These data highlight isolated mouse lymphatic vessels as a powerful new tool enabling genetic models to be applied to lymphatic physiology for the first time. (NIH R01HL089784)

Lymphatic vessels in health and disease

The lymphatic vasculature plays vital roles in tissue fluid balance, immune defense, metabolism, and cancer metastasis. In adults, lymphatic vessel formation and remodeling occur primarily during inflammation, development of the corpus luteum, wound healing, and tumor growth. Unlike the blood circulation, where unidirectional flow is sustained by the pumping actions of the heart, pumping actions intrinsic to the lymphatic vessels themselves are important drivers of lymphatic flow. This review summarizes critical components that control lymphatic physiology.