Lymph Propulsion Research Articles

Transport and Immune Functions of the Lymphatic System.

Two major functions of the lymphatic system are the reabsorption of excess interstitial fluid/protein and the coordination of immune cell interactions and trafficking. Specialized junctions between lymphatic endothelial cells optimize reabsorption. The spontaneous contractions of collecting vessels provide active lymph propulsion. One-way valves prevent backflow, and chemokine gradients direct the migration of immune cells. Specialized compartments within the lymph node facilitate antigen-immune cell interactions to produce innate and acquired immunity. Lymphatic injury and/or mutations in genes controlling vessel/valve development result in contractile/valve dysfunction, reduced immune cell trafficking and, ultimately, lymphedema. Activated CD4+ T cells produce inflammatory mediators that exacerbate these processes, potentially leading to interstitial and lymphatic vessel remodeling and negatively impacting overall function. Mouse models have advanced our knowledge of lymphatic disease, but clinical trials to reduce the impact of inflammatory mediators have yielded mixed success, implying that additional factors underlying human lymphedema are not yet understood.

Pacemaking in the lymphatic system.

Lymphatic collecting vessels exhibit spontaneous phasic contractions that are critical for lymph propulsion and tissue fluid homeostasis. This rhythmic activity is driven by action potentials conducted across the lymphatic muscle cell (LMC) layer to produce entrained contractions. The contraction frequency of a lymphatic collecting vessel displays exquisite mechanosensitivity, with a dynamic range from <1 to >20 contractions per minute. A myogenic pacemaker mechanism intrinsic to the LMCs was initially postulated to account for pressure-dependent chronotropy. Further interrogation into the cellular constituents of the lymphatic vessel wall identified non-muscle cell populations that shared some characteristics with interstitial cells of Cajal, which have pacemaker functions in the gastrointestinal and lower urinary tracts, thus raising the possibility of a non-muscle cell pacemaker. However, recent genetic knockout studies in mice support LMCs and a myogenic origin of the pacemaker activity. LMCs exhibit stochastic, but pressure-sensitive, sarcoplasmic reticulum calcium release (puffs and waves) from IP3R1 receptors, which couple to the calcium-activated chloride channel Anoctamin 1, causing depolarisation. The resulting electrical activity integrates across the highly coupled lymphatic muscle electrical syncytia through connexin 45 to modulate diastolic depolarisation. However, multiple other cation channels may also contribute to the ionic pacemaking cycle. Upon reaching threshold, a voltage-gated calcium channel-dependent action potential fires, resulting in a nearly synchronous calcium global calcium flash within the LMC layer to drive an entrained contraction. This review summarizes the key ion channels potentially responsible for the pressure-dependent chronotropy of lymphatic collecting vessels and various mechanisms of IP3R1 regulation that could contribute to frequency tuning.

Modelling Lymph Propulsion in a 3D Model of Murine Collecting Vessel with Three Lymphangions in Series

The lymphatic system maintains tissue homeostasis by transporting the excess fluid from the interstitium and ultimately returning it to the venous circulation against an adverse pressure gradient and gravitational force. The spontaneous contractions of lymphangions, the building blocks of collecting vessels, and the secondary lymphatic valves play key roles in lymph propulsion. The aim of this study was to investigate lymph propulsion in a series of three contracting lymphangions in a 3D reconstructed model segmented from micro-CT scans of the collecting lymphatics in the hind limb of mice. Computational Fluid Dynamics and Fluid–Structure Interaction were used to study the behavior of flow within the collecting vessel, as well as the behavior and deformations of the vessel wall and the poroelastic interstitium. The secondary valves were modelled as porous membranes with closed or open states depending on their permeability. A sensitivity study revealed that the parameters having the most impact on the total volume of lymph propelled by active contraction of the lymphangions were the elastic modulus of the interstitium and the permeability of the secondary valves during the open states.

Lymphatic Clearance and Pump Function.

Lymphatic vessels have an active role in draining excess interstitial fluid from organs and serving as conduits for immune cell trafficking to lymph nodes. In the central circulation, the force needed to propel blood forward is generated by the heart. In contrast, lymphatic vessels rely on intrinsic vessel contractions in combination with extrinsic forces for lymph propulsion. The intrinsic pumping features phasic contractions generated by lymphatic smooth muscle. Periodic, bicuspid valves composed of endothelial cells prevent backflow of lymph. This work provides a brief overview of lymph transport, including initial lymph formation along with cellular and molecular mechanisms controlling lymphatic vessel pumping.

In Vitro Irradiation of Lymphatic Vessels Enhances Lymphatic Pump Function

Active lymphatic pumping is critical for propulsion of lymph against a pressure gradient as well as transport of lipids and immune cells to the central venous circulation. When lymphatic pumping is impaired, diminished lymph flow leads to interstitial edema, immune cell accumulation and sustained inflammation. However, a majority of radiation biology research has primarily focused on the effects of ionizing radiation (IR) on blood vessels. These studies reported that IR induces imbalance between vaso‐dilatory and ‐constrictive pathways and impairs vascular tone. Only a handful of the studies have investigated the acute effects of IR on lymphatic vessels. Furthermore, the studies evaluating IR‐induced changes in lymphatic tone and pumping in vivo are limited because, in addition to the intrinsic contractility of the lymphatic muscle, lymphatic tone and pumping in vivo is modulated by a variety of substances present in the luminal lymph and the surrounding interstitial fluid. Therefore, the goal of the present study was to characterize the effects of IR on lymphatic tone and pumping independent of the confounding effects of IR on the other tissues.Mesenteric lymphatic vessels (MLVs), isolated from male Sprague Dawley rats, were randomly divided into SHAM and IR groups and were placed in chilled APSS. MLVs from the IR group were then irradiated with X‐rays at 10 Gray Dose. Immediately after irradiation, MLVs from both groups were placed in chilled APSS until further use. MLVs were then cannulated and perfused with warm APSS. Lymphatic pumping was assessed in spontaneously contracting vessels. Role of nitric oxide (NO) and cyclooxygenase (COX) pathways was evaluated by assessing pump function in separate MLVs before and after incubation in N(ω)‐nitro‐L‐arginine methyl ester (L‐NAME; 100 µM) and Indomethacin (INDO; 10 µM).Evaluation of the lymphatic pumping in response to changes in transmural pressure revealed significantly increased contraction frequency and calculated active lymph flow in IR MLVs compared to those in SHAM MLVs. However, stroke volume normalized to passive volume and diastolic tone were not significantly different between the IR and SHAM MLVs. The increase in contraction frequency in IR MLVs was partially restored by the blockade of COX pathways with INDO. Blockade of NOS with L‐NAME, however, led to further increase in contraction frequency of IR MLVs, which was significantly higher than that in SHAM MLVs.Early studies investigating the IR effects on lymphatic endothelial cells reported that lymphatic endothelium is resistant to IR. Conflicting findings of the in vivo lymphatic studies have further confounded the insights into the lymphatic responses to IR. It has been widely believed that lymphatic vessels are unaffected by IR, and therefore, the role of the lymphatic system‐ the other half of the circulatory system, in radiation injury has been mostly ignored. However, it is evident from the findings of the present study that lymphatic vessels are indeed affected by IR. Furthermore, IR‐induced increase in contraction frequency observed in the present study suggests that lymphatic acute responses to IR differ from the blood vessel responses.

Lymphatic Collecting Vessel: New Perspectives on Mechanisms of Contractile Regulation and Potential Lymphatic Contractile Pathways to Target in Obesity and Metabolic Diseases.

Obesity and metabolic syndrome pose a significant risk for developing cardiovascular disease and remain a critical healthcare challenge. Given the lymphatic system’s role as a nexus for lipid absorption, immune cell trafficking, interstitial fluid and macromolecule homeostasis maintenance, the impact of obesity and metabolic disease on lymphatic function is a burgeoning field in lymphatic research. Work over the past decade has progressed from the association of an obese phenotype with Prox1 haploinsufficiency and the identification of obesity as a risk factor for lymphedema to consistent findings of lymphatic collecting vessel dysfunction across multiple metabolic disease models and organisms and characterization of obesity-induced lymphedema in the morbidly obese. Critically, recent findings have suggested that restoration of lymphatic function can also ameliorate obesity and insulin resistance, positing lymphatic targeted therapies as relevant pharmacological interventions. There remain, however, significant gaps in our understanding of lymphatic collecting vessel function, particularly the mechanisms that regulate the spontaneous contractile activity required for active lymph propulsion and lymph return in humans. In this article, we will review the current findings on lymphatic architecture and collecting vessel function, including recent advances in the ionic basis of lymphatic muscle contractile activity. We will then discuss lymphatic dysfunction observed with metabolic disruption and potential pathways to target with pharmacological approaches to improve lymphatic collecting vessel function.

Lymph Node Subcapsular Sinus Microenvironment-On-A-ChipModeling Shear Flow Relevant to Lymphatic Metastasis and Immune Cell Homing.

SummaryA lymph node sinus-on-a-chip adhesion microfluidic platform that recapitulates the hydrodynamic microenvironment of the lymph node subcapsular sinus was engineered. This device was used to interrogate the effects of lymph node remodeling on cellular adhesion in fluid flow relevant to lymphatic metastasis. Wall shear stress levels analytically estimated and modeled after quiescent and diseased/inflamed lymph nodes were experimentally recapitulated using a flow-based microfluidic perfusion system to assess the effects of physiological flow fields on human metastatic cancer cell adhesion. Results suggest that both altered fluid flow profiles and presentation of adhesive ligands, which are predicted to manifest within the lymph node subcapsular sinus as a result of inflammation-induced remodeling, and the presence of lymph-borne monocytic cells may synergistically contribute to the dynamic extent of cell adhesion in flow relevant to lymph node invasion by cancer and monocytic immune cells during lymphatic metastasis.

Degradation of lymphatic anatomy and function in early venous insufficiency

ObjectiveWe used near-infrared fluorescence lymphatic imaging in a pilot study to assess the lymphatics in preulcerative (C2-C4) venous insufficiency and determine whether involvement and/or degradation of lymphatic anatomy or function could play a role in the progression of chronic venous insufficiency. We also explored the role of lymphatics in early peripheral arterial disease. MethodsAfter informed consent and intradermal injections of indocyanine green for rapid lymphatic uptake, near-infrared fluorescence lymphatic imaging was used to assess the lymphatic anatomic structure and quantify the lymphatic propulsion rates in subjects with early venous insufficiency. The anatomic observations included interstitial backflow, characterized by the abnormal spreading of indocyanine green from the injection site primarily into the surrounding interstitial tissues; dermal backflow, characterized by the retrograde movement of dye-laden lymph from collecting lymphatics into the lymphatic capillaries; and lymphatic vessel segmentation and dilation. ResultsTen subjects with venous insufficiency were enrolled, resulting in two legs with C2 disease, nine legs with C3 disease, eight legs with C4 disease, and one leg with C5 disease. Interstitial and/or dermal backflow were observed in 25%, 33%, and 41% of the injection sites in each limb with C2, C3, and C4 disease, respectively. Distinct vessel segmentation and dilation were observed in limbs with a C3 and higher classification, and dermal backflow proximal to the injection sites was observed in two legs with C4 disease and in the inguinal region of the C5 study subject. The overall average lymph propulsion rates were 1.3 ± 0.4, 1.2 ± 0.7, and 0.8 ± 0.5 contractile events/min for limbs with C2, C3, and C4 disease, respectively. One subject with peripheral arterial disease, who had previously undergone bypass surgery, presented with extensive dermal backflow and lymphatic reflux. ConclusionsNear-infrared fluorescence lymphatic imaging demonstrated that, compared with normal health subjects, the lymphatic anatomy and contractile function generally degrade with the severity of venous insufficiency. Lymphatic abnormalities mimic those in early cancer-acquired lymphedema subjects, as previously observed by us and others. Additional studies are needed to decipher the relationship, including any causality, between lymphatic dysfunction and peripheral vascular disease and venous insufficiency.

What Is the Mechanism Underlying Pressure‐induced Chronotropy in Collecting Lymphatic Vessels?

The rhythmic contractions of lymphatic smooth muscle (LSM) cells enable lymph propulsion against adverse pressure gradients. These contractions are initiated by LSM action potentials (APs), generated by an ionic pacemaker, whose rate is sensitive to the transmural pressure difference. We recently showed that SM‐specific deletion of the Ca2+‐activated Cl− channel, anoctamin1 (Ano1), reduced the basal AP firing rate of mouse inguinal‐axillary lymphatic (IALs) vessels to ~25% of normal and abrogated pressure‐induced chronotopy. However, IALs are efferent vessels that show only a modest (2‐fold) increase in contraction frequency over the presumed physiological pressure range 0.5 – 8 cmH2O. In contrast, popliteal afferent lymphatics (PLs) exhibit an 8‐fold increase in chronotropy from 0.5 – 5 cmH2O and SM‐specific deletion of Ano1 attenuates but does not abolish their pressure‐induced chronotropy. Here we asked: 1) what other ionic mechanisms participate in pressure‐induced chronotropy of PLs? 2) what accounts for the residual, pressure‐independent pacemaking in IALs and PLs after Ano1 deletion?To address these questions we measured the frequency‐pressure relationship (F‐P) of cannulated, pressurized PLs from control mice and mice deficient in key mechanosensitive and second‐messenger‐gated ion channels implicated in arterial pressure sensing. SM‐specific deletion of the mechanosensitive cation channel Piezo1 had no significant effect on F‐P. Global deletion of the osmosensitive cation channel TRPV4, or the DAG‐gated cation channels TRPC3 or TRPC6, or both, had no significant effect on F‐P. TRPM4−/− mice were not available, but the selective TRPM4 inhibitors, CBA and NBA, had minimal effects on F‐P. SM‐specific deletion of the cation channel TRPP1 (Pkd2) had no significant effect on F‐P. Deletion of T‐type Ca2+ channels Cav3.1 and/or Cav3.2 (previously implicated in lymphatic pacemaking), had no significant effect on F‐P. However, SM‐specific deletion of IP3R1, the only IP3R isoform expressed in mouse LSM, produced a phenotype similar to Ano1 deletion: a reduction in basal frequency and blunting of the F‐P relationship, suggesting a pressure‐induced IP3 generation downstream from mechanosensitive G‐protein(s). We then tested the prominent arterial SM mechanosensitive GPCR, AT1R. The inverse agonist losartan had no effect on F‐P nor did global knock out of AT1bR, the only isoform expressed in mouse LSM. In conclusion, our results point to a GPCR mechanosensing mechanism, independent of AT1R, that generates IP3 in a pressure‐sensitive manner to activate Ano1 and regulate the cycling rate of the ionic pacemaker in lymphatic smooth muscle.Support or Funding InformationNIH‐HL‐122578

Acidosis inhibits rhythmic contractions of human thoracic ducts.

Lymph vessels counteract edema by transporting interstitial fluid from peripheral tissues to the large veins and serve as conduits for immune cells, cancer cells, and pathogens. Because edema during inflammation and malignancies is frequently associated with acidosis, we tested the hypothesis that acid‐base disturbances affect human thoracic duct contractions. We studied, by isometric and isobaric myography, the contractile function of human thoracic duct segments harvested with written informed consent from patients undergoing esophageal cancer surgery. Human thoracic ducts produce complex contractile patterns consisting of tonic rises in tension (isometric myography) or decreases in diameter (isobaric myography) with superimposed phasic contractions. Active tone development decreases substantially (~90% at 30 vs. 7 mmHg) at elevated transmural pressure. Acidosis inhibits spontaneous as well as noradrenaline‐ and serotonin‐induced phasic contractions of human thoracic ducts by 70–90% at extracellular pH 6.8 compared to 7.4 with less pronounced effects observed at pH 7.1. Mean tension responses to noradrenaline and serotonin – averaged over the entire period of agonist exposure – decrease by ~50% at extracellular pH 6.8. Elevating extracellular [K+] from the normal resting level around 4 mmol/L increases overall tension development but reduces phasic activity to a level that is no different between human thoracic duct segments investigated at normal and low extracellular pH. In conclusion, we show that extracellular acidosis inhibits human thoracic duct contractions with more pronounced effects on phasic than tonic contractions. We propose that reduced phasic activity of lymph vessels at low pH attenuates lymph propulsion and increases the risk of edema formation.

Calcium and electrical dynamics in lymphatic endothelium.

Endothelial cell function in resistance arteries integrates Ca2+ signalling with hyperpolarization to promote relaxation of smooth muscle cells and increase tissue blood flow. Whether complementary signalling occurs in lymphatic endothelium is unknown. Intracellular calcium and membrane potential were evaluated in endothelial cell tubes freshly isolated from mouse collecting lymphatic vessels of the popliteal fossa. Resting membrane potential measured using intracellular microelectrodes averaged ∼-70mV. Stimulation of lymphatic endothelium by acetylcholine or a TRPV4 channel agonist increased intracellular Ca2+ with robust depolarization. Findings from Trpv4-/- mice and with computational modelling suggest that the initial mobilization of intracellular Ca2+ leads to influx of Ca2+ and Na+ through TRPV4 channels to evoke depolarization. Lymphatic endothelial cells lack the Ca2+ -activated K+ channels present in arterial endothelium to generate endothelium-derived hyperpolarization. Absence of this signalling pathway with effective depolarization may promote rapid conduction of contraction along lymphatic muscle during lymph propulsion. Subsequent to a rise in intracellular Ca2+ ([Ca2+ ]i ), hyperpolarization of the endothelium coordinates vascular smooth muscle relaxation along resistance arteries during blood flow control. In the lymphatic vasculature, collecting vessels generate rapid contractions coordinated along lymphangions to propel lymph, but the underlying signalling pathways are unknown. We tested the hypothesis that lymphatic endothelial cells (LECs) exhibit Ca2+ and electrical signalling properties that facilitate lymph propulsion. To study electrical and intracellular Ca2+ signalling dynamics in lymphatic endothelium, we excised collecting lymphatic vessels from the popliteal fossa of mice and removed their muscle cells to isolate intact LEC tubes (LECTs). Intracellular recording revealed a resting membrane potential of ∼-70mV. Acetylcholine (ACh) increased [Ca2+ ]i with a time course similar to that observed in endothelium of resistance arteries (i.e. rapid initial peak with a sustained 'plateau'). In striking contrast to the endothelium-derived hyperpolarization (EDH) characteristic of arteries, LECs depolarized (>15mV) to either ACh or TRPV4 channel activation. This depolarization was facilitated by the absence of Ca2+ -activated K+ (KCa ) channels as confirmed with PCR, persisted in the absence of extracellular Ca2+ , was abolished by LaCl3 and was attenuated ∼70% in LECTs from Trpv4-/- mice. Computational modelling of ion fluxes in LECs indicated that omitting K+ channels supports our experimental results. These findings reveal novel signalling events in LECs, which are devoid of the KCa activity abundant in arterial endothelium. Absence of EDH with effective depolarization of LECs may promote the rapid conduction of contraction waves along lymphatic muscle during lymph propulsion.

A Validation Study of Near-Infrared Fluorescence Imaging of Lymphatic Vessels in Humans

Near-infrared fluorescence (NIRF) imaging is a new imaging technique that is used to visualize lymphatic vessels in humans. It has a high spatial and temporal resolution, allowing real-time visualization of lymphatic flow. The current study investigated the intra- and inter-individual variability of the technique, and how local hypo- and hyperthermia (20°C, 40°C), as well as exercise affect lymph transport. In this study, 10 healthy volunteers were studied twice, with 2 weeks between. NIRF imaging was conducted by using intradermal Indocyanine green injections and a custom-built camera setup. All data were blinded before analysis and presented as mean ± standard deviation. Mean contraction frequency and lymph propulsion velocity were 0.59 ± 0.13 minutes-1 and 1.51 ± 0.24 cm/s, respectively, with no significant difference during each 4 hours examination or between the two visits. The maximal pressure that the lymphatic flow in the vessels could overcome on test day 1 and 2 was 56 ± 9 mmHg and 57 ± 9 mmHg, respectively (p = 0.496). Local hyperthermia increased contraction frequency from 0.62 ± 0.4 minutes to 1.46 ± 0.5 minutes-1 (p < 0.05). Hypothermia caused no significant changes. Immediately after exercise (exercising at a simulated distance of 1.4 km on a cycle ergometer), an increase in lymph propulsion velocity from 1.5 ± 0.49 to 2.2 ± 0.63 cm/s was observed (p < 0.05); whereas contraction frequency was unaltered. A decrease in contraction frequency from 0.68 ± 0.25 minutes to 0.35 ± 0.19 minutes-1 was observed 10 minutes after exercise, without a change in velocity. NIRF imaging can be conducted for 4 hours without a change in lymphatic activity. Furthermore, it has the sensitivity to detect changes in lymphatic activity by local hyperthermia and exercise. No changes were seen after local hypothermia. Pumping pressure shows good repeatability, whereas the other parameters show poor repeatability.

Fine structure of human thoracic duct as revealed by light and scanning electron microscopy.

Little information has been available regarding microanatomy of human thoracic duct in spite of the importance for an understanding of pathophysiology in clinical medicine. The present study demonstrated a fine structure of human thoracic duct system by light and scanning electron microscopy. A number of longitudinal or spiral ridges and grooves were formed on luminal surfaces of the lymphangia and lymph sac, it likely facilitating fluent lymph flow. The endothelial cells displayed various cell shapes in compliance with their distributed regions. The lymph sac joining large vein composed a peculiar multiple valve structure presumably ensuring lymph storage and prevention of lymph backflow. The longitudinal muscle sheet in the tunica intima and circular muscle bundles in the tunica media constructed an integrated power unit probably eliciting spontaneous lymph propulsion. Furthermore, the thoracic duct was richly supplied with blood vessels not only in the tunica externa, but also just beneath the endothelium. The present findings provide a morphological basis for investigation of human thoracic duct in basic and clinical medicine.

Lymphatic pumping: mechanics, mechanisms and malfunction.

A combination of extrinsic (passive) and intrinsic (active) forces move lymph against a hydrostatic pressure gradient in most regions of the body. The effectiveness of the lymph pump system impacts not only interstitial fluid balance but other aspects of overall homeostasis. This review focuses on the mechanisms that regulate the intrinsic, active contractions of collecting lymphatic vessels in relation to their ability to actively transport lymph. Lymph propulsion requires not only robust contractions of lymphatic muscle cells, but contraction waves that are synchronized over the length of a lymphangion as well as properly functioning intraluminal valves. Normal lymphatic pump function is determined by the intrinsic properties of lymphatic muscle and the regulation of pumping by lymphatic preload, afterload, spontaneous contraction rate, contractility and neural influences. Lymphatic contractile dysfunction, barrier dysfunction and valve defects are common themes among pathologies that directly involve the lymphatic system, such as inherited and acquired forms of lymphoedema, and pathologies that indirectly involve the lymphatic system, such as inflammation, obesity and metabolic syndrome, and inflammatory bowel disease.

Impact of Dynamic Transmural Pressure on Lymph Propulsion in Lymphatic Vessels

Transmural pressure is an important determinant of pumping in collecting lymphatic vessels. Here we studied the impact of dynamic transmural pressure in lymphatic vessels using our physiology‐based computational model [1]. In parallel, we conducted in vitro studies in isolated lymphatic vessels from rat mesentery to examine the effect of dynamic transmural pressure. Vessel segments containing multiple valves were isolated, cannulated, and pressurized at varying levels of inlet and outlet pressures (Pin = Pout, zero transaxial pressure difference). Dynamic pressure waves at different physiologic frequencies (0 – 0.5 Hz) and amplitudes (0 – Pin) were introduced at the inlet and outlet reservoirs. Intraluminal pressure (measured with a servo‐null system) and inner diameter were recorded throughout the experiment. At all pressure amplitudes and frequencies (both higher and lower than normal contraction frequency of the vessel), the dynamic pressure wave was superimposed on the physiologic contraction of the vessel and was detectable in both the intraluminal pressure and diameter traces. The results of our computational model demonstrated that dynamic transmural pressure increased the flow rate at low baseline transmural pressures. Under these circumstances increasing the amplitude and/or frequency of oscillations further facilitated the pumping. Additionally, our computational model enables us to control the transmural pressure by varying external pressure (independent of inlet and outlet pressures). This allows us to study conditions with non‐zero transaxial pressure differences. For example, for an adverse pressure difference of 3 cmH2O and average transmural pressure of 3 cmH2O, average flow rate increased 10% when transmural pressure oscillated with an amplitude of 1 cmH2O; doubling the contraction frequency resulted in a further 15% higher flow rate. These results suggest that dynamic transmural pressure can play an important role in maintaining lymph propulsion in areas of weaker intrinsic pumping activity.Support or Funding InformationGrant support: NIH R01 HL094269, U01 HL123420 (JEM), NIH R01 HL‐120867 (MJD). JEM wishes to acknowledge the support of The Royal Society, The Royal Academy of Engineering, and The Sir Leon Bagrit Trust.

Lymphaticosclerosis: a new way of thinking about lymphatic vessel obstruction.

ORIGINAL ARTICLE, p 1286 Lymphoedema is a progressive condition that often manifests following cancer surgeries that involve removal or irradiation of lymph nodes. The symptoms may range from mild to severe and debilitating, and in the latter case can significantly increase susceptibility to infection and restrict quality of life.1 2 It is clear that removal of lymph nodes or severing of lymphatic vessels can cause an immediate interruption of the normal flow of lymph from the upstream vessel network into the downstream collecting vessels.3 However, the pathological mechanisms that progressively lead to severe secondary (acquired) lymphoedema are unclear, as are the reasons why different individuals receiving the same procedure may exhibit wide‐ranging severities of lymphatic dysfunction. In this issue of the BJD, Ogata et al.4 have investigated the change in phenotype of lymphatic smooth muscle cells during lymphoedema. The authors studied lymphatic vessels from 29 randomly chosen patients who had developed secondary leg lymphoedema after surgical removal of tumours, were refractory to congenital conservative therapy and received a lymphaticovenous anastomosis procedure. Typically, the collecting lymphatics that are responsible for pumping of lymph in the limbs have a thin layer of circular smooth muscle cells, which intrinsically contracts. The phasic contractions of these cells, in concert with intraluminal one‐way valves composed of endothelial cells and connective tissue, allow forward propulsion of lymph through the system.5 Ogata et al.4 observed a striking change in the collecting lymphatic wall in patients with chronic lymphoedema, with a markedly thickened smooth muscle layer, increased collagen deposition and apparent invasion of smooth muscle cells into the intimal layer. In cases of more severe lymphoedema, stenosis or ‘lymphaticosclerosis’ of the lymphatic lumen due to overgrowth of lymphatic smooth muscle cells was apparent. In addition, labelling of the myosin heavy chain isoforms SM1 and SM2 was reduced in lymphatic smooth muscle, and transmission electron microscopy revealed an increase in synthetic organelles, such as sarcoplasmic reticulum and Golgi complex, while contractile myofilaments were reduced.4 Collectively, these findings suggest that the progression of surgery‐induced lymphatic injury into lymphoedema involves re‐entry of lymphatic smooth muscle cells into a proliferative stage to respond to the injury, followed by their failure to mature and remodel appropriately. This change in the phenotype of lymphatic smooth muscle is akin to a common problem with angioplasty: restenosis of the lumen after plaque removal due to the synthetic vascular smooth muscle phenotype.6

Abstract 2052: Assessing lymphatic response to treatments in head and neck cancer using near-infrared fluorescence imaging

Abstract Radiation toxicity is a major obstacle to effective head and neck (HN) cancer treatment. Treatment-related toxicity to lymphatic tissues in the HN region could significantly diminish patient's quality-of-life, with lymphedema resulting in more than half of HN cancer patients who undergo surgery and radiotherapy. Impairment of lymphatics due to radiotherapy may be expected because radiation exposures could cause tissue fibrosis and loss of capillary lymphatics. In this pilot study, we sought to visualize lymphatic architecture and contractile function before and after cancer treatments using near-infrared fluorescence (NIRF) lymphatic imaging. The approach allowed us to longitudinally assess the effect of radiation or surgery on the lymphatics in HN cancer patients. Human subjects, who were diagnosed with HN squamous cell carcinoma and scheduled for radiotherapy or combination of surgery/radiotherapy, were recruited in this ongoing study under FDA approval (IND 102,765). Imaging sessions are conducted before and as late as one year after the treatments at several time points. After informed consent, ten injections of 12.5-25 mcg indocyanine green (off-label use) in 0.05-0.1 c.c. saline were each administered bilaterally in the oral cavity, oropharynx, and on the face (posterior auricular, preauricular, and submandibular) in each imaging session. Immediately after injections, the skin surface was illuminated with a diffused excitation light of 785 nm, and sequences of NIRF lymphatic images of 0.2 sec exposure time at 2 frames/sec were collected using a custom-built imaging system. Imaging was typically performed for an hour. While subject recruitment continues, NIRF images in all 13 subjects show the lymphatic draining vessels and nodes in the face and neck. Images acquired before treatment, post-surgery, and then post-radiation allowed us to document the change of lymphatics and investigate the response of lymphatics to the treatments. Propulsion of lymph in collecting lymphatic vessels were seen in some of subjects both pre- and post- surgery as well as after radiotherapy. To date, no major impairment of lymphatics has been noted in the first 13 subjects. Longitudinal imaging was accomplished for 3 out of 13 subjects. Extra-lymphatic leakage around the incision line was seen in some subjects post surgery. In one subject, lymphatic propulsion was seen before treatment and post- radiation, but not post-surgery. Preliminary results show the reformation of lymphatic architecture after surgery and radiotherapy. In conclusion, we have employed NIRF imaging to assess lymphatics in HN cancer patients, who underwent radiotherapy and/or surgery, and to visualize the response to the treatments. While longitudinal studies continue, the preliminary results show the reorganization of lymphatics after the treatments. Citation Format: I-Chih Tan, Ron J. Karni, John C. Rasmussen, Eva M. Sevick-Muraca. Assessing lymphatic response to treatments in head and neck cancer using near-infrared fluorescence imaging. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2052. doi:10.1158/1538-7445.AM2014-2052

The contribution of K(+) channels to human thoracic duct contractility.

In smooth muscle cells, K(+) permeability is high, and this highly influences the resting membrane potential. Lymph propulsion is dependent on phasic contractions generated by smooth muscle cells of lymphatic vessels, and it is likely that K(+) channels play a critical role in regulating contractility in this tissue. The aim of this study was to investigate the contribution of distinct K(+) channels to human lymphatic vessel contractility. Thoracic ducts were harvested from 43 patients and mounted in a wire myograph for isometric force measurements or membrane potential recordings with an intracellular microelectrode. Using K(+) channel blockers and activators, we demonstrate a functional contribution to human lymphatic vessel contractility from all the major classes of K(+) channels [ATP-sensitive K(+) (KATP), Ca(2+)-activated K(+), inward rectifier K(+), and voltage-dependent K(+) channels], and this was confirmed at the mRNA level. Contraction amplitude, frequency, and baseline tension were altered depending on which channel was blocked or activated. Microelectrode impalements of lymphatic vessels determined an average resting membrane potential of -43.1 ± 3.7 mV. We observed that membrane potential changes of <5 mV could have large functional effects with contraction frequencies increasing threefold. In general, KATP channels appeared to be constitutively open since incubation with glibenclamide increased contraction frequency in spontaneously active vessels and depolarized and initiated contractions in previously quiescent vessels. The largest change in membrane voltage was observed with the KATP opener pinacidil, which caused 24 ± 3 mV hyperpolarization. We conclude that K(+) channels are important modulators of human lymphatic contractility.

KCNQ and ERG channels control the rate of diastolic depolarization and electrical pacemaking frequency in lymphatic muscle (666.3)

The spontaneous contractions of lymphatic muscle cells are critical for propulsion of lymph. We investigated the ionic basis of pacemaking in the muscle layer of pressurized rat mesentery and mouse popliteal lymphatics using sharp electrodes and patch clamp methods. At very low pressures (&lt;0.5 cmH2O) membrane potential was stable at ~ ‐42 mV, with infrequent action potentials (APs). At higher pressures APs fired at regular intervals, interspersed with slow diastolic depolarizations (DD) to a threshold of ~ ‐35 mV. Each AP consisted of a single 200‐ms spike, peaking at ~ +5 mV, followed by a 1.5‐sec plateau and after‐hyperpolarization to ~ ‐45 mV. Graded increases in pressure to 5 cmH2O led to graded increases in DD slope and AP frequency. The KCNQ inhibitors XE991 and linopirdine (1‐10 μM) increased DD slope without significantly changing AP height or duration. The selective Kv7.1 activator flupirtine (10 μM) decreased DD slope and AP frequency. In contrast, the ERG‐1 inhibitor E‐4031 (2‐6 μM) had little effect on frequency but prolonged AP duration due to multiple spikes with each AP. Based on PCR, the predominant KCNQ channel isoform was Kv7.1, with lesser expression of Kv7.4 (but not 7.2, 3 or 5). ERG‐1a and 1b (but not ERG‐2 or 3) channels were detected. We conclude that these K+ channel isoforms, which are typical of cardiac nodal cells, are important regulators of electrical pacemaking in lymphatic muscle.Grant Funding Source: NIH‐HL089784

Spontaneous activity in peripheral diaphragmatic lymphatic loops

The spontaneous contractility of FITC-dextran-filled lymphatics at the periphery of the pleural diaphragm was documented for the first time "in vivo" in anesthetized Wistar rats. We found that lymphatic segments could be divided into four phenotypes: 1) active, displaying rhythmic spontaneous contractions (51.8% of 197 analyzed sites); 2) stretch-activated, whose contraction was triggered by passive distension of the vessel lumen (4.1%); 3) passive, which displayed a completely passive distension (4.5%); and 4) inert, whose diameter never changed over time (39.6%). Smooth muscle actin was detected by immunofluorescence and confocal microscopy in the vessel walls of active but also of inert sites, albeit with a very different structure within the vessel wall. Indeed, while in active segments, actin was arranged in a dense mesh completely surrounding the lumen, in inert segments actin decorated the vessels wall in sparse longitudinal strips. When located nearby along the same lymphatic loop, active, stretch-activated, and passive sites were always recruited in temporal sequence starting from the active contraction. The time delay was ∼0.35 s between active and stretch-activated and 0.54 s between stretch-activated and passive segments, promoting a uniform lymph flux of ∼150/200 pl/min. We conclude that, unlike more central diaphragmatic lymphatic vessels, loops located at the extreme diaphragmatic periphery do require an intrinsic pumping mechanism to propel lymph centripetally, and that such an active lymph propulsion is attained by means of a complex interplay among sites whose properties differ but are indeed able to organize lymph flux in an ordered fashion.