A novel role of Hippo-Yap/TAZ signaling pathway in lymphatic vascular development

The lymphatic vasculature plays important role in regulating fluid homeostasis, intestinal lipid absorption, and immune surveillance in humans. Malfunction of lymphatic vasculature leads to several human diseases. Understanding the fundamental mechanism in lymphatic vascular development not only expand our knowledge, but also provide a new therapeutic insight. Recently, Hippo-YAP/TAZ signaling pathway, a key mechanism of organ size and tissue homeostasis, has emerged as a critical player that regulate lymphatic specification, sprouting, and maturation. In this review, we discuss the mechanistic regulation and pathophysiological significant of Hippo pathway in lymphatic vascular development.     

Mucosal lymphatic-derived gammadelta T cells respond early to experimental Salmonella enterocolitis by increasing expression of IL-2R alpha

To better understand the roles of gammadelta T cells in mucosal infection, we utilized Salmonella enterica serovar Typhimurium (Salmonella serovar Typhimurium) infection in cattle as it closely approximates Salmonella serovar Typhimurium-induced enterocolitis in humans. Protein and gene expression in alphabeta and gammadelta T cells derived from lymphatic ducts draining the gut mucosa in Salmonella serovar Typhimurium-infected calves were analyzed. In calves with enterocolitis, general gene expression trends in gammadelta T cells suggested subtle activation and innate response, whereas alphabeta T cells were relatively quiescent following Salmonella serovar Typhimurium infection. An increase in IL-2R alpha expression on gammadelta T cells from infected calves and results from in vitro assays suggested that gammadelta T cells were primed by Salmonella serovar Typhimurium LPS to better respond to IL-2 and IL-15. Together with gene expression trends in vivo, these data support early priming activation of target tissue gammadelta T cells during Salmonella serovar Typhimurium infection.     

Expression of cys-cys chemokine ligand 21 on human gingival lymphatic vessels

The cys-cys (C-C) chemokine ligand 21 is a member of the C-C chemokines that constitute a group of heparin-binding cytokines with a pattern of four or six conserved cysteines. The CCL21 is known to be expressed in secondary lymphoid tissues, however it has rarely been reported for the expression on peripheral lymphatic vessels in somatic tissue. Here we investigated the expression of CCL21 on lymphatic vessels identified by anti-desmoplakin in uninflamed and inflamed human gingiva. In uninflamed tissue the expression of CCL21 was detected on lymphatic vessels in gingiva. In uninflamed gingiva the expression of CCL21 was detected on all lymphatic capillaries of the mucosal connective tissue papillae. There were two types of collecting lymphatic vessels in the lamina propria mucosae expressing CCL21 strongly or very weakly. In inflamed gingiva no expression of CCL21 was detected on lymphatic vessels. In all tissue sections no blood vessels expressing CCL21 were observed. These results may suggest that the expression of CCL21 is predominantly induced in the peripheral lymphatic endothelium of the uninflamed mucosal microcirculation, and that under inflamed conditions a reduction of CCL21 occurs in lymphatic endothelium.     

自发性高血压大鼠肠系膜微静脉白细胞-内皮细胞相互作用和微淋巴管收缩特性的研究

目的:探讨自发性高血压大鼠(Spontaneously Hypertensive Rat,SHR)肠系膜微静脉白细胞-内皮细胞相互作用和微淋巴管收缩的特性。方法:取8周龄Wistar大鼠、8周龄SHR(SHR8W)和13周龄SHR(SHR13W),麻醉、固定并暴露肠系膜后,微循环显微镜下观察肠系膜微循环并录像;回放录像,计算微静脉白细胞滚动数和滚动的白细胞-内皮细胞接触时间(Rolling leukocyte-endothelial contact time,RLECT),用Vas Track自动测量系统对微淋巴管口径进行动态测量,并计算微淋巴管收缩特性指标。结果:SHR13W的白细胞滚动数显著低于Wistar;SHR8W和SHR13W的RLECT均显著低于Wistar,且SHR13W的RLECT显著低于SHR8W;进一步按照管径分级后,三组间白细胞滚动数在10~20μm管径级别下未见差异;各个管径级别下,SHR8W和SHR13W的RLECT均未见差异。SHR13W的淋巴管收缩分数显著低于Wistar和SHR8W;SHR8W及SHR13W的总收缩活性指数均显著低于Wistar;SHR13W的淋巴管动力指数显著低于Wistar。结论:SHR肠系膜微静脉白细胞滚动数及RLECT减少,其中白细胞滚动数在不同管径级别微静脉中的分布不均匀,而RLECT随SHR周龄降低,意味着SHR淋巴管收缩功能降低。     

Differential proteomic analysis of lymphatic,venous, and arterial endothelial cells extracted from bovine mesenteric vessels

Differential protein profiling by 2‐D PAGE is generally useful in biomarker discovery, proteome analysis and routine sample preparation prior to analysis by MS. The goal of this study was to compare 2‐D PAGE‐resolved protein profile of lymphatic endothelial cells to those of venous, and arterial endothelial cells isolated from lymphatic and blood vessels of bovine mesentery (bm). Three 2‐D PAGE electrophoretograms were produced for each of the three cell types and quantitatively analyzed. Protein identification by LC‐MS/MS was performed to identify 39 proteins found to be present at statistically significantly different levels in the three cell types (p<0.05). Most of the 39 proteins have not been previously reported in EC proteomic studies of 2‐D PAGE electrophoretograms. Three proteins, HSPA1B (HSP70 family member), HSPB1 (HSP27 family member), and UBE2D3 (a member of E2 ubiquitin‐conjugating enzymes) found to be at highest levels in bm arterial endothelial cells, bm venous endothelial cells, and bm lymphatic endothelial cells, respectively, were validated by immunoblotting with appropriate antibodies. The lack of substantial overlap between our results and those of other groups' comparative studies are discussed. Functional implications of differences in levels of various proteins identified in the three cell types are also discussed.     

Localisation of lymphatic vessels and vascular endothelial growth factors-C and -D in human and mouse skeletal muscle with immunohistochemistry

The present study was aimed to localise lymphatic vessels and their growth factors in human and mouse skeletal muscle with immunohistochemistry and specific antibodies (VEGFR-3, LYVE-1, VEGF-C and VEGF-D). The largest lymphatic vessels were found in perimysial connective tissue next to the arteries and veins, as has been shown earlier with electron microscopy. As a new finding, we also found small LYVE-1 positive vessels in the capillary bed between muscle fibres. These vessels were located next to CD31 positive blood capillaries and were of the same size, but fewer in number. In addition, we described the localisation of the two main lymphangiogenic growth factor proteins, vascular endothelial growth factor-C and -D. Both proteins were expressed in skeletal muscle at mRNA and protein levels. VEGF-D was located under the sarcolemma in some of the muscle fibres, in the endothelia of larger blood vessels and in fibroblasts. VEGF-C protein was localised to the nerves and muscle spindles, to fibroblasts and surrounding connective tissue, but was not found in muscle fibres or endothelial cells. Our results are the first to suggest the presence of lymphatic capillaries throughout the skeletal muscle, and to present the localisation of VEGF-C and -D in the muscles. Electronic Supplementary Material Supplementary material is available to authorised users in the online version of this article at .     

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.     

Molecular characterization of a truncated antigen (Wb14) of SXP-1 of Wuchereria bancrofti from four endemic regions in India

Wb14 of Wuchereria bancrofti, an orthologue of Brugia malayiSXP-1 and W. bancrofti SXP-1, was amplified from genomic DNA of W. bancrofti microfilaria collected from four distant geographical locations in India viz., Vellore, Bhubaneshwar, Pondicherry and Sevagram. The gene was sub-cloned in a prokaryotic vector pRSET and expressed in Escherichia coli as a truncated protein (∼23 kDa). The nucleotide sequence of the gene is 98% similar to that of WbSXP-1 and is found to be intron-less. However, the analysis and comparison of the derived amino acid sequence with WbSXP-1 showed that Wb14 is truncated at amino acid position 153. The distribution of the two genes in the studied four geographical locations indicated that WbSXP-1 is prevalent only in parasite samples from Sevagram while Wb14 is present in parasites from all the other locations. Only a limited polymorphism was observed in both the genes among the parasites from different geographical locations.     

胃癌组织中幽门螺杆菌L型感染与淋巴管形成关系的研究

目的 研究胃癌组织中D2-40、LYVE-1标记的微淋巴管密度(LVD)、血管内皮生长因子受体(VEGFR-3)表达与幽门螺杆菌L型(helicobacter pylori L-form,Hp-L型)感染之间的关系.方法 应用革兰染色和免疫组化SP法检测80例胃癌组织和25例对照组的Hp-L型感染,同时用免疫组化SP法检测上述组织的LVD值和VEGFR-3的表达,分析Hp-L型与LVD以及VEGFR-3表达的关系结果 胃癌组织中革兰染色L型检出阳性率为67.5％;免疫组化Hp-L型抗原表达阳性率为65％,两种方法检测同时阳性的病例50例,占62.5％.胃癌组的Hp-L型阳性率、LVD及VEGFR-3表达阳性率均高于对照组(P＜0.01);胃癌组中Hp-L阳性组的LVD值和VEGFR-3表达阳性率高于Hp-L阴性组.LVD与胃癌淋巴结转移具有一定关系.结论 Hp-L型感染与胃癌的发生、发展密切相关,Hp-L型可能是肿瘤淋巴管生成的重要促进因子,影响胃癌的侵袭和转移.     

Clinical and pathological aspects of filarial lymphedema and its management

Lymphatic filariasis, transmitted by mosquitoes is the commonest cause of lymphedema in endemic countries. Among 120 million infected people in 83 countries, up to 16 million have lymphedema. Microfilariae ingested by mosquitoes grow into infective larvae. These larvae entering humans after infected mosquito bites grow in the lymphatics to adult worms that cause damage to lymphatics resulting in dilatation of lymph vessels. This earliest pathology is demonstrated in adults as well as in children, by ultrasonography, lymphoscintigraphy and histopathology studies. Once established, this damage was thought to be irreversible. This lymphatic damage predisposes to bacterial infection that causes recurrent acute attacks of dermato-lymphangio-adenitis in the affected limbs. Bacteria, mainly streptococci gain entry into the lymphatics through 'entry lesions' in skin, like interdigital fungal infections, injuries, eczema or similar causes that disrupt integrity of skin. Attacks of dermato-lymphangio-adenitis aggravates lymphatic damage causing lymphedema, which gets worse with repeated acute attacks. Elephantiasis is a late manifestation of lymphatic filariasis, which apart from limbs may involve genitalia or breasts. Lymphedema management includes use of antifilarial drugs in early stages, treatment and prevention of acute attacks through 'limb-hygiene', antibiotics and antifungals where indicated, and physical measures to reduce the swelling. In selected cases surgery is helpful.