The Lymphatic Vascular System: Does Nonuniform Lymphangion Length Limit Flow-Rate?

A previously developed model of a lymphatic vessel as a chain of lymphangions was investigated to determine whether lymphangions of unequal length reduce pumping relative to a similar chain of equal-length ones. The model incorporates passive elastic and active contractile properties taken from ex vivo measurements, and intravascular lymphatic valves as transvalvular pressure-dependent resistances to flow with hysteresis and transmural pressure-dependent bias to the open state as observed experimentally. Coordination of lymphangion contractions is managed by marrying an autonomous transmural pressure-dependent pacemaker for each lymphangion with bidirectional transmission of activation signals between lymphangions, qualitatively matching empirical observations. With eight lymphangions as used here and many nonlinear constraints, the model is capable of complex outcomes. The expected flow-rate advantage conferred by longer lymphangions everywhere was confirmed. However, the anticipated advantage of uniform lymphangions over those of unequal length, compared in chains of equal overall length, was not found. A wide variety of dynamical outcomes was observed, with the most powerful determinant being the adverse pressure difference, rather than the arrangement of long and short lymphangions. This work suggests that the wide variation in lymphangion length which is commonly observed in collecting lymphatic vessels does not confer disadvantage in pumping lymph.

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

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

A 1D model characterizing the role of spatiotemporal contraction distributions on lymph transport.

Lymphedema is a condition in which lymph transport is compromised. The factors that govern the timing of lymphatic contractions are largely unknown; however, these factors likely play a central role in lymphatic health. Computational models have proven useful in quantifying changes in lymph transport; nevertheless, there is still much unknown regarding the regulation of contractions. The purpose of this paper is to utilize computational modeling to examine the role of pacemaking activity in lymph transport. A 1D fluid-solid modeling framework was utilized to describe the interaction between the contracting vessel and the lymph flow. The distribution of contractions along a three-lymphangion chain in time and space was determined by specifying the pacemaking sites and parameters obtained from experimentation. The model effectively replicates the contractility patterns in experiments. Quantitatively, the flow rates were measured at 5.44 and 2.29 [Formula: see text], and the EF values were 78% and less than 33% in the WT and KO models, respectively, which are consistent with the literature. Applying pacemaking parameters in this modeling framework effectively captures lymphatic contractile wave propagations and their relation to lymph transport. It can serve as a motivation for conducting novel studies to evaluate lymphatic pumping function during the development of lymphedema.

Phytocorrection of Age-Related Changes in the Composition of Blood Plasma, Lymph, and Interstitial Fluid.

We studied the effect of a new complex phytocomposition on age-related changes in the composition of biological fluids (blood, lymph, and interstitial fluid) in old (22-24-month-old) white Sprague-Dawley rats. A 3-month course of phytocomposition induced an increase in the volumes of interstitial fluid (IF) and plasma and stimulated lymph outflow and diuresis. In the blood and lymph, the clotting time increased, while the viscosity and all lipid indicators decreased. The phytocomposition increased the number of erythrocytes and leukocytes, the levels of immunoglobulins (except IgG) and lymphocyte subpopulations, which contributed to correction and improvement of the immune properties of the blood and lymph. Increased hydration of the tissues of the body and strengthening of the anti-atherogenic and immune properties of the lymph and blood led to recovery of the drainage and detoxification function of the lymphatic system. Due to the presence of bioflavonoids, microelements, and vitamins, the new complex phytocomposition produces a lymphotropic effect by changing the composition of the blood, lymph, and IF and stimulates fluid transport from IF into the vascular bed, thereby promoting natural lymph detoxification and increasing the immune properties of the blood and lymph.

Utilization of endogenous albumin trafficking pathways in the lungs has potential to modestly increase the lung interstitial access and absorption of drug delivery systems after inhaled administration.

OBJECTIVES: Drug delivery systems typically show limited access to the lung interstitium and absorption after pulmonary delivery. The aim of this work was to undertake a proof-of-concept investigation into the potential of employing endogenous albumin and albumin absorption mechanisms in the lungs to improve lung interstitial access and absorption of inhaled drug delivery systems that bind albumin. METHODS: The permeability of human albumin (HSA) through monolayers of primary human alveolar epithelia, small airway epithelia, and microvascular endothelium were investigated. The pulmonary pharmacokinetics of bovine serum albumin (BSA) was also investigated in efferent caudal mediastinal lymph duct-cannulated sheep after inhaled aerosol administration. RESULTS: Membrane permeability coefficient values (Papp) of HSA increased in the order alveolar epithelia

Chronic alcohol feeding alters lymph and plasma proteome in a rodent model.

Chronic alcohol consumption in rodents induces mesenteric collecting lymphatic vessel hyperpermeability, lymph leakage, and consequent immunometabolic dysregulation of the perilymphatic adipose tissue (PLAT). The specific lymphatic components mediating PLAT immunometabolic dysregulation remain to be identified. Specifically, whether alcohol impacts lymph composition is unknown. This study aimed to determine alcohol associated changes in lymph and plasma proteome. Adult male rats were fed a Lieber-DeCarli liquid diet containing 36 % of calories from alcohol for 10 weeks. Time-matched control animals were pair-fed. At sacrifice lymph was collected for 2 h using the lymph-fistula technique and plasma was collected prior to sacrifice. Quantitative discovery-based proteomics identified a total of 703 proteins. An integrative approach combining Ingenuity Pathway Analysis (IPA) and an unbiased network analysis using WGCNA (Weighted Gene Co-expression Network Analysis) was used to analyze the proteomics data. IPA results identified significant upregulation of a cluster of apolipoproteins in lymph from alcohol-fed animals compared with pair-fed controls and a downregulation of 34 proteins in the plasma from alcohol-fed animals. WGCNA analysis identified several candidate hub proteins in the lymph that were also significantly differentially expressed in lymph from alcohol-fed animals compared to that of pair-fed controls. WGCNA analysis of plasma identified a module without significant enrichment of differentially expressed proteins. Of the 59 proteins contained within this module, only 2 were significantly differentially expressed in plasma from alcohol-fed rats compared to plasma of pair-fed controls. Future studies will investigate further the functionality of the hub proteins affected by alcohol feeding in both lymph and plasma.

[Obesity and secondary lymphedema]. / Adipositas und sekundäres Lymphödem.

Obesity causes all types of lymphedema to deteriorate. Obesity-associated lymphedema is by now the most frequent secondary lymphedema and constitutes an entity in its own right. Obesity and its comorbidities, due to mechanical and inflammatory effects, decrease lymphatic transport and create a vicious circle of lymph stasis, local adipogenesis, and fibrosis. The therapeutic strategy must therefore address both lymphedema and obesity and its comorbidities.

Cell surface ATP synthase-released H+ and ATP play key roles in cocoa butter intake-mediated regulation of gut immunity through releases of cytokines in rat.

Proper food intake is important for maintaining good health in humans. Chocolate is known to exert anti-inflammatory effects; however, the mechanisms remain unclear. In this study, we aimed to investigate the effects of cocoa butter intake on gut immunity in rats and rabbits. Cocoa butter intake increased the lymph flow, cell density, and IL-1ß, IL-6 and IL-10 levels in mesenteric lymph. Clodronate, a macrophage depletion compound, significantly enhanced the release of all cytokines. The immunoreactivities of macrophage markers CD68 and F4/80 in the jejunal villi were significantly decreased with clodronate. Piceatannol, a selective cell surface ATP synthase inhibitor significantly reduced the cocoa butter intake-mediated releases of IL-1ß, IL-6 and IL-10. The immunoreactivities of cell surface ATP synthase were observed in rat jejunal villi. Shear stress stimulation on the myofibroblast cells isolated from rat jejunum released ATP and carbon dioxide depended with H+ release. In rabbit in vivo experiments, cocoa butter intake increased the concentrations of ATP and H+ in the portal vein. The in vitro experiments with isolated cells of rat jejunal lamina propria the pH of 3.0 and 5.0 in the medium released significantly IL-1ß and IL-6. ATP selectively released IL-10. These findings suggest that cocoa butter intake regulates the gut immunity through the release and transport of IL-1ß, IL-6, and IL-10 into mesenteric lymph vessels in a negative feedback system. In addition, the H+ and ATP released from cell surface ATP synthase in jejunal villi play key roles in the cocoa butter intake-mediated regulation of gut immunity.

Augmented experimental design for bioavailability enhancement: a robust formulation of abiraterone acetate.

Abiraterone acetate (ABRTA) is clinically beneficial in management of metastatic castration-resistant prostate cancer (PC-3). With highlighted low solubility and permeability, orally hampered treatment of ABRTA necessitate high dose to achieve therapeutic efficacy. To triumph these challenges, we aimed to develop intestinal lymphatic transport facilitating lipid-based delivery to enhance bioavailability. ABRTA-containing self-nano emulsified drug delivery (ABRTA-SNEDDS) was statistically optimized by D-optimal design using design expert. Optimized formulation was characterized for particle size, thermodynamic stability, in vitro release, in vivo bioavailability, intestinal lymphatic transport, in vitro cytotoxic effect, anti-metastatic activity, and apoptosis study. Moreover, hemolysis and histopathology studies have been performed to assess pre-clinical safety. Nano-sized particles and successful saturated drug loading were obtained for optimized formulation. In vitro release upto 98.61 ± 3.20% reveal effective release of formulation at intestinal pH 6.8. ABRTA-SNEDDS formulation shows enhanced in vivo exposure of Abiraterone (2.5-fold) than ABRTA suspension in Sprague-Dawley rats. In vitro efficacy in PC-3 cell line indicates 3.69-fold higher therapeutic potential of nano drug delivery system. Hemolysis and histopathology study indicates no significant toxicities to red blood cells and tissues, respectively. Apparently, an opportunistic strategy to increasing bioavailability of ABRTA via intestinal lymphatic transport will create a viable platform in rapidly evolving chemotherapy. Enhanced translational utility of delivery was also supported through in vitro therapeutic efficacy and safety assessments. HighlightsAbiraterone acetate is a prostate cancer drug, impeded with low bioavailability.ABRTA loaded in self nano emulsifying drug delivery enhanced its bioavailability.Intestinal lymphatic transport played role in enhanced bioavailability of ABRTA.ABRTA-SNEDDS enhanced in vitro cytotoxic activity of ABRTA.ABRTA-SNEDDS found safe in preclinical safety evaluations.

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.

The Isolation of Flowing Mesenteric Lymph in Mice to Quantify In Vivo Kinetics of Dietary Lipid Absorption and Chylomicron Secretion.

Intestinal lipoproteins, especially triglyceride-rich chylomicrons, are a major driver of metabolism, inflammation, and cardiovascular diseases. However, isolating intestinal lipoproteins is very difficult in vivo because they are first secreted from the small intestine into the mesenteric lymphatics. Chylomicron-containing lymph then empties into the subclavian vein from the thoracic duct to deliver components of the meal to the heart, lungs, and, ultimately, whole-body circulation. Isolating naïve chylomicrons is impossible from blood since chylomicron triglyceride undergoes hydrolysis immediately upon interaction with lipoprotein lipase and other lipoprotein receptors in circulation. Therefore, the original 2-day lymph fistula procedure, described by Bollman et al. in rats, has historically been used to isolate fresh mesenteric lymph before its entry into the thoracic vein. That protocol has been improved upon and professionalized by the laboratory of Patrick Tso for the last 45 years, allowing for the analysis of these critical lipoproteins and secretions from the gut. The Tso lymph fistula procedure has now been updated and is presented here visually for the first time. This revised procedure is a single-day surgical technique for installing a duodenal feeding tube, cannulating the mesenteric lymph duct, and collecting lymph after a meal in conscious mice. The major benefits of these new techniques include the ability to reproducibly collect lymph from mice (which harnesses the power of genetic mouse models); the reduced anesthesia time for mice during the implantation of the duodenal infusion tube and the lymph cannula; the ability to continuously sample lymph throughout the feeding and post-prandial period; the ability to quantitatively measure hormones and cytokines before their dilution and enzymatic hydrolysis in blood; and the ability to collect large quantities of lymph for isolating intestinal lipoproteins. This technique is a powerful tool for directly and quantitatively measuring dietary nutrient absorption, intestinal lipoprotein synthesis, and chylomicron secretion.

Correlation of Cytokine Content in the Lymph with the Morphological Parameters of the Mesenteric Lymph Nodes in Adjuvant Therapy of Experimental Breast Cancer with the Use of Fragmented Human DNA.

We studied the relationship between the level of cytokines in the lymph of the thoracic duct and the morphometric parameters of the mesenteric lymph nodes after surgical treatment of breast cancer, chemotherapy, and administration of fragmented (double-stranded, dsDNA) human DNA. In comparison with surgical treatment and with chemotherapy alone, administration of a human dsDNA has a stimulating effect on the T-cell link of the immune response. In the paracortical zone, the relationship between the chemokine MCP-1 and increased content of small lymphocytes in this zone was revealed. Interrelations of IL-2 cytokines with small lymphocytes and of IL-4 with medium lymphocytes were revealed in germinal centers. We also observed interrelations of IL-7 with small lymphocytes and IL-4 with macrophages in the medullary cords, chemokine MIP-1α with immature and mature plasma cells (the number of these cells is reduced), and of MCP-1 with immunoblasts (the number of which is also reduced) in the medullary sinuses.

A single-day mouse mesenteric lymph surgery in mice: an updated approach to study dietary lipid absorption, chylomicron secretion, and lymphocyte dynamics.

The intestine plays a crucial role in regulating whole-body lipid metabolism through its unique function of absorbing dietary fat. In the small intestine, absorptive epithelial cells emulsify hydrophobic dietary triglycerides (TAGs) prior to secreting them into mesenteric lymphatic vessels as chylomicrons. Except for short- and medium-chain fatty acids, which are directly absorbed from the intestinal lumen into portal vasculature, the only way for an animal to absorb dietary TAG is through the chylomicron/mesenteric lymphatic pathway. Isolating intestinal lipoproteins, including chylomicrons, is extremely difficult in vivo because of the dilution of postprandial lymph in the peripheral blood. In addition, once postprandial lymph enters the circulation, chylomicron TAGs are rapidly hydrolyzed. To enhance isolation of large quantities of pure postprandial chylomicrons, we have modified the Tso group's highly reproducible gold-standard double-cannulation technique in rats to enable single-day surgery and lymph collection in mice. Our technique has a significantly higher survival rate than the traditional 2-day surgical model and allows for the collection of greater than 400 µl of chylous lymph with high postprandial TAG concentrations. Using this approach, we show that after an intraduodenal lipid bolus, the mesenteric lymph contains naïve CD4+ T-cell populations that can be quantified by flow cytometry. In conclusion, this experimental approach represents a quantitative tool for determining dietary lipid absorption, intestinal lipoprotein dynamics, and mesenteric immunity. Our model may also be a powerful tool for studies of antigens, the microbiome, pharmacokinetics, and dietary compound absorption.

Enteral glucose, absorbed and metabolized, potently enhances mesenteric lymph flow in chow- and high-fat-fed rats.

A portion of absorbed dietary triglycerides (TG) is retained in the intestine after the postprandial period, within intracellular and extracellular compartments. This pool of TG can be mobilized in response to several stimuli, including oral glucose. The objective of this study was to determine whether oral glucose must be absorbed and metabolized to mobilize TG in rats and whether high-fat feeding, a model of insulin resistance, alters the lipid mobilization response to glucose. Lymph flow, TG concentration, TG output, and apolipoprotein B48 (apoB48) concentration and output were assessed after an intraduodenal lipid bolus in rats exposed to the following intraduodenal administrations 5 h later: saline (placebo), glucose, 2-deoxyglucose (2-DG, absorbed but not metabolized), or glucose + phlorizin (intestinal glucose absorption inhibitor). Glucose alone, but not 2-DG or glucose + phlorizin treatments, stimulated lymph flow, TG output, and apoB48 output compared with placebo. The effects of glucose in high-fat-fed rats were similar to those in chow-fed rats. In conclusion, glucose must be both absorbed and metabolized to enhance lymph flow and intestinal lipid mobilization. This effect is qualitatively and quantitatively similar in high-fat- and chow-fed rats. The precise signaling mechanism whereby enteral glucose enhances lymph flow and mobilizes enteral lipid remains to be determined.NEW & NOTEWORTHY Glucose potently enhances mesenteric lymph flow in chow- and high-fat-fed rats. The magnitude of glucose effect on lymph flow is no different in chow- and high-fat-fed rats. Glucose must be absorbed and metabolized to enhance lymph flow and mobilize intestinal lipid.

Lymph-derived chemokines direct early neutrophil infiltration in the lymph nodes upon Staphylococcus aureus skin infection.

A large number of neutrophils infiltrate the lymph node (LN) within 4 h after Staphylococcus aureus skin infection (4 h postinfection [hpi]) and prevent systemic S. aureus dissemination. It is not clear how infection in the skin can remotely and effectively recruit neutrophils to the LN. Here, we found that lymphatic vessel occlusion substantially reduced neutrophil recruitment to the LN. Lymphatic vessels effectively transported bacteria and proinflammatory chemokines (i.e., Chemokine [C-X-C motif] motif 1 [CXCL1] and CXCL2) to the LN. However, in the absence of lymph flow, S. aureus alone in the LN was insufficient to recruit neutrophils to the LN at 4 hpi. Instead, lymph flow facilitated the earliest neutrophil recruitment to the LN by delivering chemokines (i.e., CXCL1, CXCL2) from the site of infection. Lymphatic dysfunction is often found during inflammation. During oxazolone (OX)-induced skin inflammation, CXCL1/2 in the LN was reduced after infection. The interrupted LN conduits further disrupted the flow of lymph and impeded its communication with high endothelial venules (HEVs), resulting in impaired neutrophil migration. The impaired neutrophil interaction with bacteria contributed to persistent infection in the LN. Our studies showed that both the flow of lymph from lymphatic vessels to the LN and the distribution of lymph in the LN are critical to ensure optimal neutrophil migration and timely innate immune protection in S. aureus infection.

Subcutaneous delivery of a dendrimer-BH3 mimetic improves lymphatic uptake and survival in lymphoma.

As a malignant tumour of lymphatic origin, B-cell lymphoma represents a significant challenge for drug delivery, where effective therapies must access malignant cells in the blood, organs and lymphatics while avoiding off-target toxicity. Subcutaneous (SC) administration of nanomedicines allows preferential access to both the lymphatic and blood systems and may therefore provide a route to enhanced drug exposure to lymphomas. Here we examine the impact of SC dosing on lymphatic exposure, pharmacokinetics (PK), and efficacy of AZD0466, a small molecule dual Bcl-2/Bcl-xL inhibitor conjugated to a 'DEP®' G5 poly-l-lysine dendrimer. PK studies reveal that the plasma half-life of the dendrimer-drug conjugate is 8-times longer than that of drug alone, providing evidence of slow release from the circulating dendrimer nanocarrier. The SC dosed construct also shows preferential lymphatic transport, with over 50% of the bioavailable dose recovered in thoracic lymph. Increases in dose (up to 400 mg/kg) are well tolerated after SC administration and studies in a model of disseminated lymphoma in mice show that high dose SC treatment outperforms IV administration using doses that lead to similar total plasma exposure (lower peak concentrations but extended exposure after SC). These data show that the DEP® dendrimer can act as a circulating drug depot accessing both the lymphatic and blood circulatory systems. SC administration improves lymphatic exposure and facilitates higher dose administration due to improved tolerability. Higher dose SC administration also results in improved efficacy, suggesting that drug delivery systems that access both plasma and lymph hold significant potential for the treatment of haematological cancers where lymphatic and extranodal dissemination are poor prognostic factors.

Spontaneous contractions of the human thoracic duct-Important for securing lymphatic return during positive pressure ventilation?

The thoracic duct is responsible for the circulatory return of most lymphatic fluid. The return is a well-timed synergy between the pressure in the thoracic duct, venous pressure at the thoracic duct outlet, and intrathoracic pressures during respiration. However, little is known about the forces determining thoracic duct pressure and how these respond to mechanical ventilation. We aimed to assess human thoracic duct pressure and identify elements affecting it during positive pressure ventilation and a brief ventilatory pause. The study examined pressures of 35 patients with severe congenital heart defects undergoing lymphatic interventions. Thoracic duct pressure and central venous pressure were measured in 25 patients during mechanical ventilation and in ten patients during both ventilation and a short pause in ventilation. TD contractions, mechanical ventilation, and arterial pulsations influenced the thoracic duct pressure. The mean pressure of the thoracic duct was 16 ± 5 mmHg. The frequency of the contractions was 5 ± 1 min-1 resulting in an average increase in pressure of 4 ± 4 mmHg. During mechanical ventilation, the thoracic duct pressure correlated closely to the central venous pressure. TD contractions were able to increase thoracic duct pressure by 25%. With thoracic duct pressure correlating closely to the central venous pressure, this intrinsic force may be an important factor in securing a successful return of lymphatic fluid. Future studies are needed to examine the return of lymphatic fluid and the function of the thoracic duct in the absence of both lymphatic complications and mechanical ventilation.

Gravity and lymphodynamics.

The lymphatic system is compromised in different groups of patients. To recognize pathology, we must know what is healthy. We use Near-Infrared Fluorescence (NIRF) to assess peripheral lymphatic function in humans. We have shown that external factors such as exercise, hyperthermia, and pharmacological mediators influence the function of peripheral lymphatic vessels. In this study, we explored the impact on lymphatic vessels by the ever-present external factor-gravity. We used NIRF imaging to investigate the lymphatic changes to gravity. Gravity was assessed by changing body position from supine to standing. We extracted following lymphatic functional parameters: lymphatic packet propulsion frequency (contractions/min), velocity (cm/s), and pressure (mmHg). Raw data analysis was performed using a custom-written Labview program. All sequences were analyzed by two observers and interclass correlation scores were calculated. All statistical analysis was performed using RStudio Team (2021). RStudio: Integrated Development Environment for R. RStudio, PBC. Healthy participants (n = 17, 11 males, age 28.1 ± 2.6 years) were included. The lymphatic packet propulsion frequency at baseline was 0.5 ± 0.2 contractions/min and rose within 3 min significantly to a maximum of 1.2 ± 0.5 contractions/min during upright posture and remained significantly higher than the baseline lymphatic packet propulsion frequency after lying down again for up to 6 min. The lymph velocity was 1.5 ± 0.4 cm/s at baseline and changed in both directions and without a specific pattern at different points in time during standing. Lymph pressure was significantly higher while standing (mean increase 9 mmHg, CI: 2-15 mmHg). The ICC scores were 89.8% (85.9%-92.7%), 59.3% (46.6%-69.6%) and 89.4% (79.0%-94.8%) in lymphatic packet propulsion frequency (130 observations), velocity (125 observations), and pressure (30 observations), respectively. The lymphatic system responds within few minutes to gravitational changes by increasing lymphatic packet propulsion frequency and pressure.