Collecting Lymph Vessels Research Articles

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

Acute Exposure of Collecting Lymphatic Vessels to Low-Density Lipoproteins Increases Both Contraction Frequency and Lymph Flow: AnIn VivoMechanical Insight

Background: Lymphatic vessels drain fluids and solutes from interstitial spaces and serosal cavities. Among the solutes, low-density lipoproteins (LDL) are drained and can be detected in peripheral lymph, where they have been reported to exert a modulatory action on lymphatic vessels intrinsic contraction rate. In the present work, we investigated lymphatic vessel mechanical properties (contraction frequency and amplitude) that may be modulated by LDL application and the consequence on lymph flow. Methods and Results: Human-derived LDL were resuspended in phosphate-buffered saline (PBS) and microinjected in the interstitial space surrounding spontaneously contracting lymphatic vessels of the rat diaphragm, in vivo. Vessels' contraction rate and diameter were measured in control conditions (PBS) and after LDL injection. Lymph flow (Jlymph) was computed from contraction rate and diameter change. In some animals, after the recording procedure, diaphragmatic tissue samples were excised and immunostained with antilymphatic muscle (LM) actin to investigate the correlation between LM signal level and contraction amplitude. Data indicate a positive, saturating correlation between the abundance of LM actin and contraction amplitude, and LDL microinjection caused an acute increase in contraction frequency (+126%), a reduction of contraction amplitude to 75% of that obtained after PBS injection, and a +63% increase in Jlymph. Conclusions: From our in vivo analysis of the mechanical parameters affected by LDL, Jlymph was increased by a predominant effect on the contraction rate rather than amplitude, suggesting that the still elusive messaging system might be linked to the pacemaker sites.

Doxorubicin Activates Ryanodine Receptors in Rat Lymphatic Muscle Cells to Attenuate Rhythmic Contractions and Lymph Flow.

Doxorubicin is a risk factor for secondary lymphedema in cancer patients exposed to surgery or radiation. The risk is presumed to relate to its cytotoxicity. However, the present study provides initial evidence that doxorubicin directly inhibits lymph flow and this action appears distinct from its cytotoxic activity. We used real-time edge detection to track diameter changes in isolated rat mesenteric lymph vessels. Doxorubicin (0.5-20 μmol/l) progressively constricted lymph vessels and inhibited rhythmic contractions, reducing flow to 24.2% ± 7.7% of baseline. The inhibition of rhythmic contractions by doxorubicin paralleled a tonic rise in cytosolic Ca2+ concentration in lymphatic muscle cells, which was prevented by pharmacological antagonism of ryanodine receptors. Washout of doxorubicin partially restored lymph vessel contractions, implying a pharmacological effect. Subsequently, high-speed optical imaging was used to assess the effect of doxorubicin on rat mesenteric lymph flow in vivo. Superfusion of doxorubicin (0.05-10 μmol/l) maximally reduced volumetric lymph flow to 34% ± 11.6% of baseline. Likewise, doxorubicin (10 mg/kg) administered intravenously to establish clinically achievable plasma concentrations also maximally reduced volumetric lymph flow to 40.3% ± 6.0% of initial values. Our findings reveal that doxorubicin at plasma concentrations achieved during chemotherapy opens ryanodine receptors to induce "calcium leak" from the sarcoplasmic reticulum in lymphatic muscle cells and reduces lymph flow, an event linked to lymph vessel damage and the development of lymphedema. These results infer that pharmacological block of ryanodine receptors in lymphatic smooth muscle cells may mitigate secondary lymphedema in cancer patients subjected to doxorubicin chemotherapy. SIGNIFICANCE STATEMENT: Doxorubicin directly inhibits the rhythmic contractions of collecting lymph vessels and reduces lymph flow as a possible mechanism of secondary lymphedema, which is associated with the administration of anthracycline-based chemotherapy. The inhibitory effects of doxorubicin on rhythmic contractions and flow in isolated lymph vessels were prevented by pharmacological block of ryanodine receptors, thereby identifying the ryanodine receptor family of proteins as potential therapeutic targets for the development of new antilymphedema medications.

Deep lymphatic anatomy of the upper limb: An anatomical study and clinical implications

BackgroundThe deep and perforating lymphatic anatomy of the upper limb still remains the least described in medical literature. Materials and MethodsSix upper limbs with the axillary tissue were harvested from three unembalmed human cadavers amputated at the shoulder joint. A small amount of 6% hydrogen peroxide was employed to detect the lymphatic vessels around the deep palmar arch, radial and ulnar neurovascular bundles. A 30-gauge needle was inserted into the vessels and they were injected with a barium sulphate compound. Each specimen was dissected, photographed and radiographed to demonstrate deep lymphatic distribution of the upper limb. ResultsContinuing from the deep lymph vessels of the hand, single or multiple deep collecting lymph vessels have been found along the radial, ulnar, anterior and posterior interosseous neurovascular bundles in the forearm, brachial and deep branchial neurovascular bundles in the upper arm. During their courses, lymph nodes were found setting in the trunk of the radial, ulnar and brachial lymph vessels near or in the cubital fossa, and in the axillar. Perforating lymph vessels have been found near the wrist and in the cubital fossa, which linked the superficial and deep lymph vessels. The direction of lymphatic drainage was from the deep to superficial or superficial to deep vessels. ConclusionThe deep lymphatic anatomy of the upper limb has been described. The results will provide an anatomical basis for clinical management, educational reference and scientific research.

Ultrasonography for classifying lymphatic sclerosis types and deciding optimal sites for lymphatic-venous anastomosis in patients with lymphoedema,

We have previously categorised of degeneration of the collecting lymphatic vessels into four types: normal, ectasis, contraction and sclerosis type (NECST classification). Herein, we evaluated the collecting lymphatic vessels in lymphoedema-affected limbs using ultrasonography. In step 1, we investigated 110 lymphatic vessels from 25 patients with lymphoedema, who underwent lymphatic-venous anastomosis (LVA) following preoperative ultrasonography. We classified the lymphatic vessels using the NECST classification during intraoperative microscopic observation. Post-operatively, we evaluated the preoperative ultrasonographic images and identified the lymphatic vessels. In step 2, we investigated 79 lymphatic vessels from 17 patients. We performed ultrasonography and detected the lymphatic vessels preoperatively and compared the results with the intraoperative findings. This study is not blinded. In step 1, normal-type lymphatic vessels were observed as spicular and flat hypo-echoic lesions on ultrasonography. Ectasis-type lymphatic vessels appeared as a rounded hypo-echoic region and coloured on Doppler imaging once in 20-30 s. Contraction-type lymphatic vessels appeared as a small hypo-echoic region in the centre of the hyper-echoic ellipse. Sclerosis-type lymphatic vessels appeared as a hyper-echoic ellipse without lumen, similar to fibrotic tissues. In step 2, of 79 lymphatic vessels found intraoperatively, 65 (82.3%) were detected on ultrasonography and 37 (46.8%) were accurately diagnosed according to the NECST classification criteria preoperatively. All lymphatic vessels detected on ultrasonography were found intraoperatively. Collecting lymphatic vessels could be observed by ultrasonography in lymphoedema-affected limbs. Depending on the degree of collecting lymphatic vessel sclerosis-corresponding to the NECST classification-various findings such as spicular, rounded, hyper-echoic and similar to these were presented. Moreover, we can decide optimal sites for LVA preoperatively.

Spontaneous and α-adrenoceptor-induced contractility in human collecting lymphatic vessels require chloride.

Human lymphatic vessels are myogenically active and respond to sympathetic stimulation. The role of various cations in this behavior has recently been investigated, but whether the anion Cl- is essential is unclear. With ethical approval and informed consent, human thoracic duct and mesenteric lymphatic vessels were obtained from surgical patients. Spontaneous or norepinephrine-induced isometric force production from isolated vessels was measured by wire myography; the transmembrane Cl- gradient and Cl- channels were investigated by substitution of extracellular Cl- with the impermeant anion aspartate and inhibition of Cl- transport and channels with the clinical diuretics furosemide and bendroflumethiazide as well as DIDS and 5-nitro-2-(3-phenylpropylamino)benzoic acid. The molecular expression of Ca2+-activated Cl- channels was investigated by RT-PCR, and proteins were localized using immunoreactivity. Spontaneous and norepinephrine-induced contractility in human lymphatic vessels was highly abrogated after Cl- substitution with aspartate. About 100-300 µM DIDS or 5-nitro-2-(3-phenylpropylamino)benzoic acid inhibited spontaneous contractile behavior. Norepinephrine-stimulated tone was furthermore markedly abrogated by 200 µM DIDS. Furosemide lowered only spontaneous constrictions, whereas bendroflumethiazide had nonspecific inhibitory effects. Consistent expression of transmembrane member 16A [TMEM16A (anoctamin-1)] was found in both the thoracic duct and mesenteric lymphatic vessels, and immunoreactivity with different antibodies localized TMEM16A to lymphatic smooth muscle cells and interstitial cells. The significant change in contractile function observed with inhibitors and anion substitution suggests that Cl- movement over the plasma membrane of lymphatic myocytes is integral for spontaneous and α-adrenoceptor-evoked contractility in human collecting lymphatic vessels. Consistent detection and localization of TMEM16A to myocytes suggests that this channel could play a major functional role. NEW & NOTEWORTHY In this study, we report the first observations of Cl- being a critical ionic component of spontaneous and agonist-evoked contractility in human lymphatics. The most consistently expressed Ca2+-activated Cl- channel gene in the human thoracic duct and mesenteric lymphatic vessels appears to be transmembrane member 16A, suggesting that this channel plays a major role.

The deep lymphatic anatomy of the hand

BackgroundThe deep lymphatic anatomy of the hand still remains the least described in medical literature. MethodsEight hands were harvested from four nonembalmed human cadavers amputated above the wrist. A small amount of 6% hydrogen peroxide was employed to detect the lymphatic vessels around the superficial and deep palmar vascular arches, in webs from the index to little fingers, the thenar and hypothenar areas. A 30-gauge needle was inserted into the vessels and injected with a barium sulphate compound. Each specimen was dissected, photographed and radiographed to demonstrate deep lymphatic distribution of the hand. ResultsFive groups of deep collecting lymph vessels were found in the hand: superficial palmar arch lymph vessel (SPALV); deep palmar arch lymph vessel (DPALV); thenar lymph vessel (TLV); hypothenar lymph vessel (HTLV); deep finger web lymph vessel (DFWLV). Each group of vessels drained in different directions first, then all turned and ran towards the wrist in different layers. ConclusionThe deep lymphatic drainage of the hand has been presented. The results will provide an anatomical basis for clinical management, educational reference and scientific research.

Lymphatic Wall Remodeling with Systemic and Tissue‐Associated Inflammation in Obese Zucker Rats

Lymphatic vessels have an intimate relationship with adipose tissue, and a growing number of reports tie dysfunction of lymphatics to obesity. A large gap currently exists in the knowledge of the pathophysiological mechanisms involved in obesity‐induced lymphatic dysfunction. The goal of this study was to investigate the extent to which obesity gives rise to inflammation and remodeling of the wall of rat mesenteric collecting lymphatic vessels. ZUCKER‐Leprafa (obese) and ZUCKER‐Leprafa/+ (lean) male and/or female rats, in conformance with the FASEB Statement of Principles for the use of Animals in Research and Education, were used for this study. Age groups included 8, 12 and 26–32 wks. Collecting lymphatic vessels were isolated from the mesentery and were mounted onto resistance‐matched glass micropipettes to determine passive wall mechanics. These experiments were performed in a 37°C albumin physiological salt solution (APSS without Ca2+) bath with luminal pressure stepped from 1–5 cm H2O. In addition, mesenteric arcades with adipose tissue and collecting lymphatics were dissected, fixed in 4% paraformaldehyde, processed and embedded in paraffin for sectioning and immunostaining with antibodies to rat‐specific CD68 (macrophage marker) and caveolin‐1 (adipocyte marker) for the quantification of crown‐like structures (CLSs). Blood (serum) and peri‐lymphatic adipose tissue (PLAT) were also collected and analyzed with the currently available panel of 67 inflammatory biomarkers through the Rat Biomarker Testing Service by RayBiotech. The results show that lymphatic wall thickness is significantly greater in obese Zucker rats compared to age‐matched counterparts. Wall:lumin ratio (X100) and medial cross‐sectional area were also significantly greater in obese versus lean male rats at all ages. Many CLSs were observed surrounding mesenteric collecting lymphatics isolated from obese male 12 and 26–32 wk rats. Additionally, CLSs significantly increased with age. In an analyses of serum biomarkers obtained from 12‐wk old lean and obese, male and female rats, we observed that many of the markers in PLAT (IL‐6, IL‐13, adiponectin) and in serum (GM‐CSF, IL‐4, Gas1) were differentially regulated by obesity and sex. The data support the concept that obesity causes an inflammatory environment in the mesentery that is associated with lymphatic vessel wall remodeling and changes to passive wall mechanics. The data also show significant differences in the profiles of inflammatory mediators increased by obesity in male and female Zucker rats, highlighting the importance of studying both sexes.Support or Funding InformationSupported by NIH L40HL097863This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

End-to-End Lymphaticovenular Anastomosis Does Not Disturb the Contraction of Collecting Lymph Vessels

Supplemental Digital Content is available in the text.

The passive biomechanics of human pelvic collecting lymphatic vessels.

The lymphatic system has a major significance in the metastatic pathways in women’s cancers. Lymphatic pumping depends on both extrinsic and intrinsic mechanisms, and the mechanical behavior of lymphatic vessels regulates the function of the system. However, data on the mechanical properties and function of human lymphatics are lacking. Our aim is to characterize, for the first time, the passive biomechanical behavior of human collecting lymphatic vessels removed at pelvic lymph node dissection during primary debulking surgeries for epithelial ovarian cancer. Isolated vessels were cannulated and then pressurized at varying levels of applied axial stretch in a calcium-free Krebs buffer. Pressurized vessels were then imaged using multi-photon microscopy for collagen-elastin structural composition and fiber orientation. Both pressure-diameter and force-elongation responses were highly nonlinear, and axial stretching of the vessel served to decrease diameter at constant pressure. Pressure-diameter behavior for the human vessels is very similar to data from rat mesenteric vessels, though the human vessels were approximately 10× larger than those from rats. Multiphoton microscopy revealed the vessels to be composed of an inner layer of elastin with an outer layer of aligned collagen fibers. This is the first study that successfully described the passive biomechanical response and composition of human lymphatic vessels in patients with ovarian cancer. Future work should expand on this knowledge base with investigations of vessels from other anatomical locations, contractile behavior, and the implications on metastatic cell transport.

Multi‐scale Modeling of Collecting Lymphatic Vessels: From Ion Channel Activity to Lymph Transport

Exact physical, molecular and cellular mechanisms underlying the transport of the lymph within the lymphatic system is highly complicated, and for the most part is poorly understood. This proposed research outlines our recent effort in developing a multiscale computational model of collecting lymphatic vessel segments (lymphangions), in which the transport function at macroscale level can be analyzed by integrating underlying mechanisms at cellular and molecular levels. This outlined approach can provide a useful platform to study the function or dysfunction of the collecting lymphatic vessels in normal and diseased states, respectively. Detailed mathematical expressions of electrophysiological properties of major ionic channels, membrane currents and intracellular stores present in a single lymphatic smooth muscle cell (LSMC) has been derived from independent experimental studies in literature. The single LSMC model predicts the detailed dynamics of calcium and membrane potential at the whole cell level via integrating individual membrane and intracellular components. A model of the lymphangion wall is constructed via connecting LSMCs through the incorporation of homocellular gap junctions between the neighboring cells. This multicellular model has the potential to lay bare on complex intra‐ and intercellular electrical signaling and propagating ionic waves. The detailed electrophysiological model of the lymphangion wall is combined with a biomechanics and fluid dynamics model to form a mathematical model of a section of the collecting lymphatic vessel (i.e. a series of lymphangions separated by unidirectional valves). This model allows us to study the real‐time relationship between the changes in the diameter and pressure of the vessel segments and changes in the concentration of ionic species. The single LSMC model successfully predicts calcium and membrane current oscillations at physiological amplitude and frequencies without the presence of an agonist. The multicellular model suggests that the chloride current is the major contributor in synchronizing the LSMC spontaneous contractions within one lymphangion, while the presence of pressure‐sensitive channels accounts for anti‐phase contractions observed in neighboring lymphangions. This proposed model is a novel attempt that synergistically takes into account the biomechanics of the lymphatic vessel wall, fluid dynamics, and the electrophysiology of major ionic species in the cells constituting the lymphatic vessels.Support or Funding InformationSupported by National Institutes of Health grant HL121778

Characterization of internodal collecting lymphatic vessel function after surgical removal of an axillary lymph node in mice.

Secondary lymphedema is an acquired lymphatic disorder, which occurs because of damage to the lymphatic system from surgery and/or radiation therapy for cancer treatment. However, it remains unknown how post-nodal collecting lymphatic vessels (CLVs) draining to the surgical wound area change in response to lymphadenectomy. We investigated functional and architectural changes of inguinal-to-axillary internodal CLVs (ICLVs) in mice after a single axillary LN (ALN) dissection using near-infrared fluorescence imaging. Our data showed no lymph flow in the ICLVs draining from the inguinal LN (ILN) at 2 days post-surgery. External compression enabled visualization of a small segment of contractile fluorescent ICLVs, but not all the way to the axillary region. At day 6, abnormal lymphatic drainage patterns, including lateral and retrograde lymph flow via vessels branching off the ICLVs were observed, which started to disappear beginning 9 days after surgery. The administration of vascular endothelial growth factor (VEGF)-C into the wound increased resolution of altered lymphatic drainage. Lymphatic drainage from the base of the tail to the ILN did not significantly change over time. These results demonstrate that lymph flow in the CLVs is dramatically affected by a LN dissection and long-term interruption of lymph flow might cause CLV dysfunction and thus contribute to chronic lymphatic disorders.

2015 Summer Biomechanics, Bioengineering, and Biotransport Conference Student Paper Competition.

As in past years, a Student Paper Competition was held at the 2015 Summer Biomechanics, Bioengineering, and Biotransport Conference (SB3C). There were three competition levels: Ph.D., M.S., and B.S. The competition was divided into multiple technical areas to provide for a fair evaluation of student work. There were also cash awards for the top three student papers in each area. We are pleased to recognize the following awardees from the 2015 SB3C.First Place: Kevin J. Warburton, Boise State University, “Wear Simulator for Canine Total Hip Replacements.”Second Place: Brett S. Klosterhoff, Purdue University, “Quantitative Visualization of Drug Response of Breast Cancer Cells Within a Three-Dimensional Extracellular Matrix.”Third Place: McKenna Drysdale, University of Utah, “Material Property Testing of Carboxymethylated Hyaluronic Acid Hydrogel Polymer.”First Place: Nicholas A. Bianco, University of Florida, “Can Measured Muscle Synergies Reconstruct Unmeasured Muscle Excitations?”Second Place: Justin A. Jones, University of Utah, “The Temporal Change in Protein Biomarkers of Vitreous Humor Following Blast Trauma of the Visual System.”Third Place: Nithya Vijayakumar, Stanford University, “A Mechanical Model for Cortical Folding During Brain Development.”First Place: Rosamaria Tricarico, University of Florida, “Elevated Wall Shear Stress Predicts Branch Graft Failure Following Chimney Endovascular Aortic Aneurysm Repair.”Second Place: Jazmine Aira, University of South Florida, “Image-Based 3D Morphometric Analysis of the Clavicle Intramedullary Canal in Male Population.”Third Place: Daniela Velez-Rendon, University of Illinois at Chicago, “Establishing in vivo Hemodynamic Baseline in a Normotensive Rat Model.”First Place: Erica E. Morrill, Boise State University, “Accurate Prediction of Collagen Fiber Distribution Using FFT: A Validation Study.”Second Place: Meghan Sylvia, California Polytechnic State University, San Luis Obispo, CA, “Development of a Human Knee Joint Finite Element Model to Investigate Cartilage Stress During Walking in Obese and Normal Weight Adults.”Third Place: Michael J. Raymond, Rensselaer Polytechnic Institute, “Individual Cell-Based Morphological Analysis to Determine Chirality of Epithelial Morphogenesis.”First Place: Priya Nair, Arizona State University, “Hemodynamic Characterization of Different Basilar Tip Aneurysm Templates Using Computational Fluid Dynamics.”Second Place: Samira Jamalian, Imperial College London, London, UK, “Suction Effect Produced by Active Contraction of Collecting Lymphatic Vessels Facilities Lymphatic Filling.”Third Place: David Schreier, University of Wisconsin–Madison, “Increased Red Blood Cell Stiffness Increases Pulmonary Vascular Resistance and Pulmonary Arterial Pressure.”First Place: M. Owais Khan, University of Toronto, Toronto, Canada, “CFD Simulation of Transition to Turbulence for Newtonian vs. Non-Newtonian Flow Through a Stenosis.”Second Place: Michael J. Weiler, Georgia Institute of Technology, “Reduced Lymphatic Function Correlates With Disease Progression in a Novel Single Vessel Ligation Model of Lymphedema.”Third Place: Anne M. Römgens, Eindhoven University of Technology, Eindhoven, The Netherlands, “Combined Experimental and Finite Element Analysis to Determine the Diffusion Coefficient Within and Between Human Skin Layers.”First Place: Feini Qu, University of Pennsylvania, “Interstitial Cell Migration in Dense Connective Tissues Is Modulated by Matrix Microstructure and Micromechanics.”Second Place: John T. Martin, University of Pennsylvania, “In Vitro Growth Trajectory and In Vivo Implantation of Cell-Seeded and Disc-Like Angle Ply Structures for Total Disc Replacement.”Third Place: Heather A. Cirka, Worcester Polytechnic Institute, “Cells Alter Traction Force and Orientation in Response to Long-Term Cyclic Stretch.”First Place: Jenell Smith, University of Pennsylvania, “Blocking Blood-Spinal Cord Barrier Breakdown Prevents the Development of Pain Following Nerve Root Compression Injury.”Second Place: Sijia Zhang, University of Pennsylvania, “Defining Collagen Fiber Mechanics in Neuron-Collagen Constructs Under Stretch Using Integrated Experimental & Modeling Approaches.”Third Place: Christopher F. Ferrigno, Rush University, “Redistribution of Knee Loads Using Auditory Feedback From Pressure Detecting Shoe Insoles.”First Place: Spencer E. Szczesny, University of Pennsylvania, “Evidence That Interfibrillar Load Transfer in Tendon Is Supported by a Network of Small Diameter Collagen Fibrils.”Second Place: Kara E. Garcia, Washington University in St. Louis, “Regional Contraction Shapes the Three-Dimensional Morphogenesis of the Embryonic Forebrain.”Third Place: Elias R. Pavlatos, The Ohio State University, “3D Strains in Posterior Sclera Using Ultrasound Speckle Tracking.”First Place: Devika M. Varma, The City College of New York, “Thermoresponsive, Redox-Crosslinked Cellulosic Hydrogels Undergo In Situ Gelation and Restore Nucleus Pulposus Biomechanical Properties Post Nucleotomy.”Second Place: Andrew P. Baumann, University of Notre Dame, “Influence of Intracortical Porosity on the Fracture Susceptibility of Human Cortical Bone.”Third Place: Robert J. Nims, Columbia University, “A Multigenerational Collagen Damage Model Explains Engineered Cartilage Growth and Remodeling Phenomena.”

Collecting lymphatic vessel permeability facilitates adipose tissue inflammation and distribution of antigen to lymph node-homing adipose tissue dendritic cells.

Collecting lymphatic vessels (CLVs), surrounded by fat and endowed with contractile muscle and valves, transport lymph from tissues after it is absorbed into lymphatic capillaries. CLVs are not known to participate in immune responses. In this study, we observed that the inherent permeability of CLVs allowed broad distribution of lymph components within surrounding fat for uptake by adjacent macrophages and dendritic cells (DCs) that actively interacted with CLVs. Endocytosis of lymph-derived Ags by these cells supported recall T cell responses in the fat and also generated Ag-bearing DCs for emigration into adjacent lymph nodes (LNs). Enhanced recruitment of DCs to inflammation-reactive LNs significantly relied on adipose tissue DCs to maintain sufficient numbers of Ag-bearing DCs as the LN expanded. Thus, CLVs coordinate inflammation and immunity within adipose depots and foster the generation of an unexpected pool of APCs for Ag transport into the adjacent LN.

Lymphatic vascular integrity is disrupted in type 2 diabetes due to impaired nitric oxide signalling.

Lymphatic vessel dysfunction is an emerging component of metabolic diseases and can lead to tissue lipid accumulation, dyslipidaemia, and oedema. While lymph leakage has been implicated in obesity and hypercholesterolaemia, whether similar lymphatic dysfunction exists in diabetes has not been investigated. To measure the lymphatic integrity of transgenic mice, we developed a new assay that quantifies the solute permeability of murine collecting lymphatic vessels. Compared with age-matched wild-type (WT) controls, the permeability of collecting lymphatics from diabetic, leptin receptor-deficient (db/db) mice was elevated >130-fold. Augmenting nitric oxide (NO) production by suffusion of l-arginine rescued this defect. Using pharmacological tools and eNOS(-/-) mice, we found that NO increased WT lymphatic permeability, but reduced db/db lymphatic permeability. These conflicting actions of NO were reconciled by the finding that phosphodiesterase 3 (PDE3), normally inhibited by NO signalling, was active in db/db lymphatics and inhibition of this enzyme restored barrier function. In conclusion, we identified the first lymphatic vascular defect in type 2 diabetes, an enhanced permeability caused by low NO bioavailability. Further, this demonstrates that PDE3 inhibition is required to maintain lymphatic vessel integrity and represents a viable therapeutic target for lymphatic endothelial dysfunction in metabolic disease.

A high salt diet alters pressure-induced mechanical activity of the rat lymphatics with enhancement of myogenic characteristics.

The lymphatic system has become a new player for pathogenesis in salt-sensitive hypertension animals. A high salt diet (HSD) evokes accumulation of Na(+) in the skin of rodents. In response to increase in Na(+)-proteoglycan complex, infiltrated macrophages stimulate secretion of vascular endothelial growth factor (VEGF)-C. Macrophage-derived VEGF-C increases density of the dermal lymph capillaries, indicating that lymphangiogenesis is advantageous to hypertensive animals by buffering elevated blood pressure. However, the effects of a high salt diet (HSD) on changes in mechanical activity of collecting lymph vessels, which directly connect with lymph capillaries, have not yet been determined. Changes in mechanical activity of isolated collecting lymphatics in normal salt diet (NSD) and HSD rats in response to increase in intraluminal pressures were measured by video-microscopy. HSD vessels had smaller % active diameters (maximum and minimum) and higher amplitude compared with NSD vessels. The frequency of lymphatic oscillation was better maintained in HSD rats than in NSD. Lymphatic pump efficiency including stroke volume index (SVI), frequency times SVI, and amplitude times frequency in HSD rats were significantly higher than those of NSD. Thus, a HSD enhances the resistance to pressure-induced decreases in lymphatic pump efficiency. The present ex vivo study suggest that collecting lymphatics of rats enhance myogenic activity and lymphatic pump efficiency to compensate for increase in lymph flow and/or pressure after 2 weeks salt loading.

Method for the quantitative measurement of collecting lymphatic vessel contraction in mice.

Collecting lymphatic vessels are critical for the transport of lymph and its cellular contents to lymph nodes for both immune surveillance and the maintenance of tissue-fluid balance. Collecting lymphatic vessels drive lymph flow by autonomous contraction of smooth muscle cells that cover these vessels. Here we describe methods using intravital microscopy to image and quantify collecting lymphatic vessel contraction in mice. Our methods allow for the measurement of the strength of lymphatic contraction of an individual lymphangion in a mouse, which has not yet been demonstrated using other published methods. The ability to study murine collecting lymphatic vessel contraction-using the methods described here or other recently published techniques-allows the field to dissect the molecular mechanisms controlling lymphatic pumping under normal and pathological conditions using the wide variety of molecular tools and genetic models available in the mouse. We have used our methods to study lymphatic contraction in physiological and inflammatory conditions. The methods described here will facilitate the further study of lymphatic function in other pathological conditions that feature lymphatic complications.

Chronic High-Fat Diet Impairs Collecting Lymphatic Vessel Function in Mice

Lymphatic vessels play an essential role in intestinal lipid uptake, and impairment of lymphatic vessel function leads to enhanced adipose tissue accumulation in patients with lymphedema and in genetic mouse models of lymphatic dysfunction. However, the effects of obesity on lymphatic function have been poorly studied. We investigated if and how adipose tissue accumulation influences lymphatic function. Using a lymphatic specific tracer, we performed in vivo near-infrared (NIR) imaging to assess the function of collecting lymphatic vessels in mice fed normal chow or high-fat diet (HFD). Histological and whole mount analyses were performed to investigate the morphological changes in initial and the collecting lymphatic vessels. HFD was associated with impaired collecting lymphatic vessel function, as evidenced by reduced frequency of contractions and diminished response to mechanostimulation. Moreover, we found a significant negative correlation between collecting lymphatic vessel function and body weight. Whole mount analyses showed an enlargement of contractile collecting lymphatic vessels of the hind limb. In K14-VEGF-C mice, HFD resulted in a reduced spreading of the tracer within dermal lymphatic vessels. These findings indicate that adipose tissue expansion due to HFD leads to a functional impairment of the lymphatic vasculature, predominantly in collecting lymphatic vessels.

Comparison of Indocyanine Green Lymphographic Findings with the Conditions of Collecting Lymphatic Vessels of Limbs in Patients with Lymphedema

The authors investigated the relationship between findings from indocyanine green lymphography and the condition of lymphatics according to the Normal, Ectasis, Contraction, Sclerosis Type classifications observed in each area during surgery. The authors examined 43 limbs of 25 patients who had undergone lymphaticovenous anastomosis from April to July of 2012 at the Department of Plastic and Reconstructive Surgery, University of Tokyo Hospital. After the injection of indocyanine green, linear, splash, stardust, and diffuse patterns were determined. Visual findings of the collecting lymph vessels during the anastomosis at each incision site were evaluated using the Normal, Ectasis, Contraction, Sclerosis Type classifications. Lymphaticovenous anastomosis was conducted on 164 collecting lymph vessels within 43 limbs of 25 people. Normal-type lymph vessels were observed in 36, ectasis type in 43, contraction type in 52, and sclerosis type in 33. Many normal vessels were found in the linear region, but the proportion of this type declined in the more severe stardust and diffuse regions. In contrast, no sclerosis-type vessels were found in the linear region, while the proportion of this type increased with lymphedema severity. Indocyanine green lymphography findings and the Normal, Ectasis, Contraction, Sclerosis Type classification of collecting lymph vessels seem to have a relationship. An increase in the severity of lymphedema according to the indocyanine green lymphography findings indicated an increase in the stage of lymphedema according to the Normal, Ectasis, Contraction, Sclerosis Type classification. Diagnostic, IV.

Regeneration of lymphatic vessels after lymphatic‐specific photodynamic therapy is not dependent on lymphangiogenesis and tissue remodeling by myofibroblast

AbstractPhotodynamic therapy (PDT) is a clinically used therapeutic procedure based on the administration of a non‐toxic photosensitizer followed by sub‐thermal light exposure for treatment of pathologic cornea neovascularization. However, this approach does not target pre‐existing lymphatics at the cornea limbus and sustained antigen drainage can lead to rejection of allogenic cornea grafts. Our PDT protocol aim at specific destruction of lymphatic vessels leaving blood circulation intact. Collecting lymphatic vessels remain occluded for 9 days and then the drainage is restored. Interestingly, lymphatic regeneration occurs not by lymphangiogenesis and wound healing but by true, epimorphic regeneration. We found that myofibroblasts were present in the tissue even though the tissue injury was limited to the lymphatic endothelial cells of collecting lymphatics and their pericyte coverage. Even though myofibroblasts were recruited and persisted in the tissue for the whole period of lymphatic regeneration no scaring in other tissue structures was observed, i.e. remodeling of blood vessels, nerve fibers or adipocytes. Limited tissue remodeling and scarification after lymphatic‐specific PDT should alleviate potential clinical translation of this method for temporal occlusion of collecting lymphatics e.g. before cornea grafting.