Reduced expression of semaphorin 3A in osteoclasts causes lymphatic expansion in a Gorham-Stout disease (GSD) mouse model / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

Gorham-Stout disease (GSD) is a sporadic chronic disease characterized by progressive bone dissolution, absorption, and disappearance along with lymphatic vessel infiltration in bone-marrow cavities. Although the osteolytic mechanism of GSD has been widely studied, the cause of lymphatic hyperplasia in GSD is rarely investigated. In this study, by comparing the RNA expression profile of osteoclasts (OCs) with that of OC precursors (OCPs) by RNA sequencing, we identified a new factor, semaphorin 3A (Sema3A), which is an osteoprotective factor involved in the lymphatic expansion of GSD. Compared to OCPs, OCs enhanced the growth, migration, and tube formation of lymphatic endothelial cells (LECs), in which the expression of Sema3A is low compared to that in OCPs. In the presence of recombinant Sema3A, the growth, migration, and tube formation of LECs were inhibited, further confirming the inhibitory effect of Sema3A on LECs in vitro. Using an LEC-induced GSD mouse model, the effect of Sema3A was examined by injecting lentivirus-expressing Sema3A into the tibiae in vivo. We found that the overexpression of Sema3A in tibiae suppressed the expansion of LECs and alleviated bone loss, whereas the injection of lentivirus expressing Sema3A short hairpin RNA (shRNA) into the tibiae caused GSD-like phenotypes. Histological staining further demonstrated that OCs decreased and osteocalcin increased after Sema3A lentiviral treatment, compared with the control. Based on the above results, we propose that reduced Sema3A in OCs is one of the mechanisms contributing to the pathogeneses of GSD and that expressing Sema3A represents a new approach for the treatment of GSD.

Changes and effects of lymphatic vessels and lymphatic endothelial cells in lymph node metastasis of esophageal cancer / 中国胸心血管外科临床杂志

@#The lymphatic system is the main way of tumor metastasis and diffusion. Esophageal cancer is one of the typical cancers that are prone to metastasis through the lymphatic system. At present, an increasing number of studies show that the interaction between tumor cells and lymphatic endothelial cells is the first step in tumor lymphatic metastasis, but the underlying molecular mechanism is unclear. This article reviews the role and changes of tumor-related lymphatic vessels and lymphatic endothelial cells in the process of tumor lymphatic metastasis, which lays a foundation for further study of the specific molecular mechanism of esophageal cancer lymphatic metastasis and provides a new treatment direction for esophageal cancer patients.

Gallbladder cancer-associated fibroblasts promote the migration of lymphatic endothelial cells via releasing IGFBP3 / 西安交通大学学报(医学版)

【Objective】 To investigate the effects of gallbladder cancer-associated fibroblasts (CAFs) on the migration of lymphatic endothelial cells (LECs) so as to elucidate the molecular mechanisms involved. 【Methods】 The CAFs and normal fibroblasts (NFs) were extracted by enzymatic digestion, and the supernatant (CM) of CAFs and NFs was collected. The levels of IL-6, IGFBP3 and other related cytokines were detected by semi-quantitative protein factor microarray and ELISA. The expressions of α-SMA (CAFs maker) and IGFBP3 in gallbladder cancer and para-cancer tissues were detected by immunohistochemistry, and the correlation of α-SMA and IGFBP3 expressions with clinicopathological characteristics were analyzed. LECs were cultured and divided into serum-free medium group (control group), CAF-CM co-culture group, NF-CM co-culture group, IGFBP3 group, and CAF-CM+IGFBP3 inhibitor (2-Deoxy-D-glucose, 2-DG) group according to different treatment. Transwell migration assays and wound healing assays were applied to analyze the migration ability of LECs under different treatment. The expressions of E-cadherin, N-cadherin and Vimentin were detected by Western blotting. 【Results】 Protein factor microarray and ELISA showed that the concentration of IGFBP3 in CAF-CM was significantly increased, and the expression of α-SMA was significantly related to lymph node metastasis, advanced TNM stage and expression of IGFBP3. IGFBP3 secreted from CAF-CM significantly promoted LECs migration, up-regulated the expression of N-cadherin and Vimentin, and down-regulated the expression of E-cadherin. Treatment with IGFBP3 inhibitor 2-DG could reverse the effect of CAF-CM on migration of LECs and related protein expressions. 【Conclusion】 Gallbladder CAFs promote the migration of LECs via releasing IGFBP3, which affects EMT transformation.

Lymphatic vasculature in tumor metastasis and immunobiology / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

Lymphatic vessels are essential for tissue fluid homeostasis, immune cell trafficking, and intestinal lipid absorption. The lymphatics have long been recognized to serve as conduits for distant tumor dissemination. However, recent findings suggest that the regional lymphatic vasculature also shapes the immune microenvironment of the tumor mass and potentiates immunotherapy. This review discusses the role of lymphatic vessels in tumor metastasis and tumor immunity.

The application of Whole-Mount immuno fluorescence staining technique in the study of lymphatic morphology in mice / 中华整形外科杂志

Objective@#To elaborate the characteristics and advantages of Whole-Mount immune fluorescence staining by observing the lymphatic vessels of mice.@*Methods@#The ear skin tissue, the hindlimb lymphatic vessels and the mesenteric lymphatic vessels were harvested from normal C57 mice. The tissue samples were subjected to whole-tissue immunofluorescence staining.These tissue samples were fixed by paraformaldehyde, blocked by bovine serum and incubated in primary and secondary antibodies. Then, the lymphatic vessels were observed and analyzed in these samples with a confocal laser-scanning microscope.@*Results@#The capillary lymphatic vessels and lymphatic endothelial cells can be clearly showed in the ear skin. The valves and smooth muscles can be clearly showed in the hindlimb and mesenteric lymphatic vessels by Whole-Mount immunofluorescence staining.@*Conclusions@#The whole-tissue immunofluorescence staining technique can observe the external morphology of lymphatic vessels clearly and stereoscopically, and can deeply observe the internal structure of lymphatic vessels. This technique can provide more accurate study on physiology and pathology of lymphatic vessels.

Lymphatic vasculature in tumor metastasis and immunobiology / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

Lymphatic vessels are essential for tissue fluid homeostasis, immune cell trafficking, and intestinal lipid absorption. The lymphatics have long been recognized to serve as conduits for distant tumor dissemination. However, recent findings suggest that the regional lymphatic vasculature also shapes the immune microenvironment of the tumor mass and potentiates immunotherapy. This review discusses the role of lymphatic vessels in tumor metastasis and tumor immunity.

Establishment and Characterization of Human Lymphatic Endothelial Cell in Breast Cancer Patients Undergoing Sentinel Lymphadenectomy / 한국유방암학회지

Recently remarkable progress has been made in the understanding of lymphangiogenesis due to the availability of specific biomarkers. A sentinel lymph node (SLN) biopsy has already been established as a common surgical procedure, and the clinical usefulness of an SLN biopsy has been confirmed in patients with various types of cancer. In this study, a novel method has been successfully developed for the isolation of anatomically defined lymphatic endothelial cells (LECs) from human sentinel lymphatic channels during an SLN biopsy in breast cancer patients by the use of collagenase II digestion. The isolated cells were cultured in EGM-2MV media with 10% fetal bovine serum (FBS) under hypoxic conditions in an atmosphere of 5% O2, 5% CO2 and 90% N2 at 37degrees C. The cultured cells exhibited a monolayer with a cobblestone appearance. Immunofluorescence analysis using selective lymphangiogenesis markers showed expression of vascular endothelial growth factor receptor 3 (VEGFR-3), prospero homeobox 1 (Prox-1) and Podoplanin. Newly established LECs showed expressions of vascular endothelial growth factor (VEGF) family members except VEGF-D and corresponding VEGF receptors by the use of conventional RT-PCR. Treatment with VEGF-C156S (500 ng/mL) apparently induced phosphorylation of the protein kinase Akt, MAPK and JNK in human isolated LECs as determined by Western blot analysis. Peak induction of Akt, MAPK and JNK occurred at 10 to 15 minutes after incubation of the isolated LECs with VEGF-C156S. The use of the MTS cell proliferation assay showed a significant increase in the growth of human lymphatic endothelial cells with VEGF-C156S treatment. The effects of VEGF-C156S (500 ng/mL) on proliferation activity was significantly with 3% FBS condition alone (MTS score: 0.54+/-0.0104, n=3) and a 3% FBS+VEGF-C156S (MTS score: 0.572+/-0.0061, n=3) condition. The isolated and enzymatic digested method adopted for the culture of human LECs is simple and useful for the investigation of the cellular, molecular and genomic properties of LECs.

INFLUENCE OF ENDOSTATIN AND PF-4 ON LYMPHANGIOGENESIS / 解剖学报

Objective Try to discover the safe and effective inhibitors of lymphangiogenesis. Methods Lymphatic endotheial cells from pig thoracic duct were isolated and cultured. The observation of specimens by LM and EM was made. The control group, endostatin and PF-4 experimental groups were set. Methods of the scraped line and MTT were used to examine their inhibitive effect on the lymphangiogenesis. Results The P value is

EFFECTS OF ENDOTHELIAL ADHESION MOLECULE PECAM-1,ICAM-3 AND CD44 ON LYMPHANGIOGENESIS IN VITRO / 解剖学报

Objective To investigate effects of endothelial adhesion molecule platelet endothelial cell odhesion molecule-1(PECAM-1),intercellular adhesion molecule-3(ICAM-3) and CD44 on lymphangiogenesis. Methods The lymphatic endothelial cells of the canine thoracic ducts were isolated and incubated.PECAM-1,ICAM-3 and CD44 on the lymphatic endothelial cells were labeled and visualized under fluorescence microscope and confocal laser scanning microscope.The endothelial cells stimulated by TNF-? or LPS were treated with the blocking antibodies against PECAM-1, ICAM-3 and CD44 respectively.The proliferation and migration of cells were determined with cell counting. The three-dimensional collagen gel model of lymphangiogenesis was prepared, and then the tube formation was viewed. The total length and area of the tubes formed from lymphatic endothelial cells were measured and their characteristics were examined with a transmission electron microscope. Results PECAM-1, ICAM-3 and CD44 were expressed on the lymphatic endothelial cells. In the control, TNF-? and LPS groups, the percentage of the migrated cells decreased and the total length and area of the tubes reduced after PECAM-1, ICAM-3 and CD44 of the cells were blocked respectively. The proliferated cells reduced after PECAM-1 or CD44 of the cells were blocked. However, there were no significant changes in the numbers of proliferated cells when ICAM-3 of the cells was blocked. In the semi-ultrathin and ultrathin sections, the tube structures formed by lymphatic endothelial cells were similar to lymphatic capillary.Conclusion PECAM-1, ICAM-3 and CD44 are expressed on the cultured lymphatic endothelial cells. These adhesion molecules are involved in lymphangiogenesis that includes proliferation, migration and tube formation of endothelial cells.