Harvesting and cryopreservation of lymphatic endothelial cells for lymphatic tissue engineering

In order to provide a suitable source of cells for lymphatic tissue engineering, the present study was designed to investigate techniques for harvesting and cryopreservation of human dermal lymphatic endothelial cells (LECs) in vitro. The LECs were isolated from children’s foreskins and then cultured in endothelial growth medium-2 MV (EGM-2-MV) with 5% FBS. The second passage LECs were suspended in cryopreservation solution containing 40% FBS and 10% Me₂SO in EGM-2-MV, cooled to −80 °C at about 1 °C/min and stored in liquid nitrogen. Samples were thawed quickly in a 37 °C water bath, and the cryoprotectant was removed by serial elution. The membrane integrity of thawed LECs was determined by trypan blue staining exclusion, and their proliferation was evaluated using the MTT method. The expanded cells of two groups were identified using immunofluorescence staining and RT-PCR with lymphatic-specific markers such as Podoplanin and VEGFR-3. Uptake of fluorescent DiI-Ac-LDL and microtubular formation in three-dimensional cultures were used to detect the function of LECs. Flow cytometry was applied to identify cells and to measure the apoptosis rate as well. Cryopreservation resulted in a retrieval of 67 ± 4% and an intact cell rate of 80 ± 3%. The early apoptosis rate of thawed LECs (9.15 ± 0.34%) was higher than that of fresh control LECs (5.31 ± 0.23%). The growth curves of thawed LECs were similar to those of fresh LECs. The thawed LECs were propagated for at least 6-7 passages without alterations in phenotype and function. Highly purified LECs can be isolated by immunomagnetic beads from human dermis. The cryopreserved/thawed and recultivated LECs are proven to have high vitality and growth potential in vitro and may be considered suitable seed cells for lymphatic tissue engineering.     

Thoracic lymphatic disorders

Thoracic complications of lymphatic disorders can culminate in respiratory failure and death and should be considered in any patient with a lymphatic disease and clinical or radiographic evidence of chest disease. Congenital lymphatic disorders are being increasingly recognized in the adult population. The spectrum of thoracic manifestations of lymphatic disorders ranges from incidental radiographic findings to diffuse lymphatic disease with respiratory failure. This article serves to review some recent advances that allow improved diagnosis and management of thoracic lymphatic disorders. Herein, we describe their anatomical and physiologic effects, the time course of their progression, and the therapies that are currently available. The management of malignant (cancerous) lymphatic disorders of the thorax is beyond the scope of this paper.     

Effect of lymphatic outflow pressure on lymphatic albumin transport in humans.

The effects of posture on the lymphatic outflow pressure and lymphatic return of albumin were examined in 10 volunteers. Lymph flow was stimulated with a bolus infusion of isotonic saline (0.9%, 12.6 ml/kg body wt) under four separate conditions: upright rest (Up), upright rest with lower body positive pressure (LBPP), supine rest (Sup), and supine rest with lower body negative pressure (LBNP). The increase in plasma albumin content (Delta Alb) during the 2 h after bolus saline infusion was greater in Up than in LBPP: 82.9 +/- 18.5 vs. -28.4 mg/kg body wt. Delta Alb was greater in LBNP than in Sup: 92.6 vs. -22.5 +/- 18.9 mg/kg body wt (P < 0.05). The greater Delta Alb in Up and Sup with LBNP were associated with a lower estimated lymphatic outflow pressure on the basis of the difference in central venous pressure (Delta CVP). During LBPP, CVP was increased compared with Up: 3.8 +/- 1.4 vs. -1.2 +/- 1.2 mmHg. During LBNP, CVP was reduced compared with Sup: -3.0 +/- 2.2 vs. 1.7 +/- 1.0 mmHg. The translocation of protein into the vascular space after bolus saline infusion reflects lymph return of protein and is higher in Up than in Sup. Modulation of CVP with LBPP or LBNP in Up and Sup, respectively, reversed the impact of posture on lymphatic outflow pressure. Thus posture-dependent changes in lymphatic protein transport are modulated by changes in CVP through its mechanical impact on lymphatic outflow pressure.     

Molecular and cellular basis of the regulation of lymphatic contractility and lymphatic absorption

Lymphatic absorption is a highly regulated process driven by both an extrinsic mechanism (external force) and an intrinsic mechanism (lymphatic vessel contractility). The lymphatic muscle is a specialized smooth muscle with unique mechanical properties. To understand the molecular mechanism and relative contribution of smooth muscle contraction in lymphatic absorption, we analyzed mice with a smooth muscle-specific deletion of Mylk, a critical gene for smooth muscle contraction. Interestingly, the knockout mice were significantly resistant to anesthesia reagents. Upon injection in the feet with FITC-dextran, the mutant mice displayed a 2-fold delay of the absorption peak in the peripheral circulation. Examining the ear lymphatic vessels of the mutant mice revealed a reduction in the amount of fluid in the lumens of the lymphangions, suggesting an impairment of lymph formation. The Mylk-deficient lymphatic muscle exhibited a significant reduction of peristalsis and of myosin light chain phosphorylation in response to depolarization. We thus concluded that MLCK and myosin light chain phosphorylation are required for lymphatic vessel contraction. Lymphatic contractility is not an exclusive requirement for lymphatic absorption, and external force appears to be necessary for absorption.     

Distribution of diaphragmatic lymphatic stomata

In seven anesthetized rabbits we measured the size, shape, and density of lymphatic stomata on the peritoneal and pleural sides of the diaphragm. The diaphragm was fixed in situ and processed for scanning electron microscopy. Results are from 2,902 peritoneal and 3,086 pleural fields (each 1,620 microns 2) randomly chosen from the various specimens. Stomata were seen in 9% of the fields examined, and in 30% of the cases they appeared grouped in clusters with 2-14 stomata/field. Stoma density was 250 +/- 242 and 72 +/- 57 (SD) stomata/mm2 on peritoneal and pleural sides, respectively, and it was similar over the muscular and tendinous portion of the two surfaces. The maximum diameter ranged from less than 1 to approximately 30 microns, with an average value of 1.2 +/- 3.1 micron. The ratio of the maximum to the minimum diameter and the surface area averaged 2 +/- 1.4 and 0.7 +/- 2.4 micron 2, respectively. The maximum and minimum diameter and surface area values followed a lognormal frequency distribution, suggesting that stomata geometry is affected by diaphragmatic tension.     

Global mapping of lymphatic filariasis

Disease maps are becoming increasingly important in infectious disease epidemiology and control. For lymphatic filariasis, the development of such maps has been hampered in the past by the lack of data on the geographical distribution of levels of infection or disease. Here, Edwin Michael and Don Bundy present an atlas for this parasitic disease derived from a recently compiled geographical database. Focusing on mapping and analysis of case prevalence data at the global and regional levels, the authors show how mapping the geographical distribution is integral not only to assessing spatial patterns in the infection and disease distribution but also to stratifying endemic areas by infection and/or disease rate.     

一氧化氮对大鼠胸膜淋巴孔调控及淋巴吸收的影响

实验研究了一氧化氮（nitric oxide,NO）对大鼠胸膜淋巴孔的调控和胸膜腔淋巴吸收的影响。NO供体和NOS（nitric oxide synthase）抑制剂分别经腹腔给药,示踪剂（台盼蓝）胸膜腔内注射后,处死大鼠,测定血清NO和台盼蓝浓度;在扫描电镜下观察各组胸膜淋巴孔,用计算机图像处理,统计学分析。结果显示,NO供体组血清NO浓度为49.34±18.47μmol/L,淋巴孔的面积和密度分别为6.80±1.13 μm2和170.24±66.60/0.1mm2;NOS抑制剂组血清NO浓度为17.72±6.58μmol/L,淋巴孔的面积和密度分别为5.72±1.54μm2和61.71±12.73/0.1mm2。血清NO浓度与淋巴孔开放的面积和密度成正相关（P<0.05）。在胸膜腔给示踪剂后,NO供体组血清台盼蓝的浓度为74.68±33.67mg/L,与对照组比较有显著差异（P<0.05）。提示,NO可以调控胸膜淋巴孔,促进胸膜腔淋巴吸收。     

Lymphangiogenesis in vitro: Formation of lymphatic capillary-like channels from confluent monolayers of lymphatic endothelial cells

Summary Lymphatic endothelial cells grown long term in culture form lymphatic capillarylike tubes. Examination by light and transmission electron microscopy showed that these structures were closed loops composed of one to several cells connected by intercellular junction to form a luminal space. This first demonstration of lymphangiogenesis in confluent monolayer cultures of lymphatic endothelial cells (a) showed that collagen type I accelerated lymphatic capillary tube formation, whereas fibronectin and matrigel had no effect; b) provided a model to study lymphatic endothelial cell function and differentiation; and c) offered a possibility to distinguish differences between the process of lymphangiogenesis and angiogenesis by testing various factors and conditions that effect endothelial cell behavior.