• Animal Bioscience
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• v.34 no.4
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• pp.533-538
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• 2021

Objective: Pluripotent stem cell-derived lymphatic endothelial cells (LECs) show great promise in their therapeutic application in the field of regenerative medicine related to lymphatic vessels. We tested the approach of forced differentiation of mouse embryonal stem cells into LECs using biodegradable poly lactic-co-glycolic acid (PLGA) nanospheres in conjugation with growth factors (vascular endothelial growth factors [VEGF-A and VEGF-C]). Methods: We evaluated the practical use of heparin-conjugated PLGA nanoparticles (molecular weight ~15,000) in conjugation with VEGF-A/C, embryoid body (EB) formation, and LEC differentiation using immunofluorescence staining followed by quantification and quantitative real-time polymerase chain reaction analysis. Results: We showed that formation and differentiation of EB with VEGF-A/C-conjugated PLGA nanospheres, compared to direct supplementation of VEGF-A/C to the EB differentiation media, greatly improved yield of LYVE1(+) LECs. Our analyses revealed that the enhanced potential of LEC differentiation using VEGF-A/C-conjugated PLGA nanospheres was mediated by elevation of expression of the genes that are important for lymphatic vessel formation. Conclusion: Together, we not only established an improved protocol for LEC differentiation using PLGA nanospheres but also provided a platform technology for the mechanistic study of LEC development in mammals.

• Maxillofacial Plastic and Reconstructive Surgery
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• v.36 no.3
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• pp.85-93
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• 2014

Purpose: Nodal metastasis is the main prognostic factor in the patients with oral squamous cell carcinoma (OSCC). We investigated the association between tumor-associated lymphatics and OSCC characteristics. Methods: Thirty-four specimens were used for the immunohistochemical staining with the antibody for vascular endothelial growth factor (VEGF)-C, VEGF-D, VEGF receptor (VEGFR)-3, phosphorylated VEGFR-3, D2-40, and matrix metallproteinases (MMPs). We observed the distribution of the lymphangiogenic factors and quantified the degree of expression. We determined lymphatic vessel density (LVD) and lymphatic vessel dilatation with D2-40 immunostaining. We assessed the association of LVD or lymphatic vessel dilatation with tumor progression or tumor differentiation. Results: OSCC cells expressed lymphangiogenic ligands. Lymphangiogenic receptor, VEGFR-3, was expressed and activated in some tumor cells as well as in tumor-associated endothelial cells. LVD was not associated with tumor size or nodal status, but lymphatic vessel dilatation was higher in tumors with nodal metastasis, and also higher in poorly differentiated tumors. In stromal area of OSCC, MMP-1 and MMP-10 were up-regulated and the basement membrane of tumor-associated endothelial cells was destroyed by these collagenases. Conclusion: In the primary tumors with nodal metastasis, especially in poorly differentiated OSCC, tumor cells invaded the dilated lymphatic vessels via ruptured sites. MMP-1 and MMP-10 are important in the lysis of the glycocalyx inside the tumor-associated lymphatic endothelial cells.

• Molecules and Cells
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• v.37 no.7
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• pp.503-510
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• 2014

Lymphatic vessels provide essential roles in maintaining fluid homeostasis and lipid absorption. Dysfunctions of the lymphatic vessels lead to debilitating pathological conditions, collectively known as lymphedema. In addition, lymphatic vessels are a critical moderator for the onset and progression of diverse human diseases including metastatic cancer and obesity. Despite their clinical importance, there is no currently effective pharmacological therapy to regulate functions of lymphatic vessels. Recent efforts to manipulate the Vascular Endothelial Growth Factor-C (VEGFC) pathway, which is arguably the most important signaling pathway regulating lymphatic endothelial cells, to alleviate lymphedema yielded largely mixed results, necessitating identification of new targetable signaling pathways for therapeutic intervention for lymphedema. Zebrafish, a relatively new model system to investigate lymphatic biology, appears to be an ideal model to identify novel therapeutic targets for lymphatic biology. In this review, we will provide an overview of our current understanding of the lymphatic vessels in vertebrates, and discuss zebrafish as a promising in vivo model to study lymphatic vessels.

• IMMUNE NETWORK
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• v.14 no.4
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• pp.182-186
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• 2014

Lymphatic vessels are routes for leukocyte migration and fluid drainage. In addition to their passive roles in migration of leukocytes, increasing evidence indicates their active roles in immune regulation. Tissue inflammation rapidly induces lymphatic endothelial cell proliferation and chemokine production, thereby resulting in lymphangiogenesis. Furthermore, lymphatic endothelial cells induce T cell tolerance through various mechanisms. In this review, we focus on the current knowledge on how inflammatory cytokines affect lymphangiogenesis and the roles of lymphatic vessels in modulating immune responses.

• Maxillofacial Plastic and Reconstructive Surgery
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• v.32 no.1
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• pp.1-8
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• 2010

Background and Purpose: Vascular endothelial growth factor (VEGF)-C and VEGF receptor (VEGFR)-3 are involved in tumor lymphangiogenesis. Oral mucosal squamous cell carcinoma (OMSCC) preferentially metastasizes to cervical lymph nodes, so we investigated the expression and distribution of VEGFR-3 signaling proteins in OMSCC. Materials and Methods: Tissue samples of 18 OMSCC, 10 oral mucosal leukoplakia, and 3 normal oral mucosa were evaluated for expression of VEGF-C, VEGF-D, and VEGFR-3 by immunohistochemical staining. The presence of lymphatic vessels was determined using D2-40 staining, by which we also measured lymphatic vessel density (LVD). Results: 72% (13/18) and 56% (10/18) of tissue samples showed VEGF-C and VEGF-D immunopositivity in tumor cells and tumor-associated endothelial cells. VEGFR-3 was also expressed in most of OMSCC, which was up-regulated when compared with normal mucosa or with leukoplakia. Furthermore, LVD was higher in OMSCC than in leukoplakia. Conclusion: Taken together, our results suggest that autocrine activation of lymphatic endothelial cell via VEGFR-3 by VEGF-C and/or VEGF-D could be involved in progression of OMSCC. Therefore, VEGF-C/VEGFR-3 signaling pathway can be a molecular target for anti-metastatic therapy in OMSCC.

• Asian Pacific Journal of Cancer Prevention
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• v.13 no.6
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• pp.2823-2828
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• 2012

Objective: To explore a new model of in-situ xenograft lymphangiogenesis of human colonic adenocarcinomas in nude mice. Method: On the basis of establishing subcutaneous xenograft lymphangiogenesis model of human colonic adenocarcinoms, in-situ xenografts were established through the in situ growth of the HT-29 human colonic adenocarcinoma cell line in nude mice. The numbers of lymphangiogenic microvessels, the expression of lymphatic endothelial cell markers lymphatic vessel endothelial hyaloronic acid receptor-1 (LYVE-1), D2-40 and the lymphatic endothelial growth factors vascular endothelial growth factor-C (VEGF-C), -D (VEGF-D) and receptor-3 (VEGFR-3) were compared by immunohistochemical staining, Western bolt and quantitative RT-PCR in xenograft in-situ models. Results: Some microlymphatics with thin walls, large and irregular or collapsed cavities and increased LMVD, with strong positive of LYVE-1, D2-40 in immunohistochemistry, were observed, identical with the morphological characteristics of lymphatic vessels and capillaries. Expression of LYVE-1 and D2-40 proteins and mRNAs were significantly higher in xenograpfts in-situ than in the negative control group(both P<0.01). Moreover, the expression of VEGF-C, VEGF-D and VEGFR-3 proteins and mRNAs were significantly higher in xenografts in-situ (both P<0.01), in conformity with the signal regulation of the VEGF-C,-D/VEGFR-3 axis of tumor lymphangiogenesis. Conclusions: In-situ xenografts of a human colonic adenocarcinoma cell line demonstrate tumor lymphangiogenesis. This novel in-situ animal model should be useful for further studying mechanisms of lymph node metastasis, drug intervention and anti-metastasis therapy in colorectal cancer.

• Journal of the Korean Association of Oral and Maxillofacial Surgeons
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• v.38 no.3
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• pp.145-151
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• 2012

Objectives: Angiogenesis and lymphangiogenesis are correlated with tumor growth and lymph node metastasis in cases of oral squamous cell carcinoma (OSCC). Endoglin is one of the representative vascular endothelial cell markers. Podoplanin is also a representative marker used in order to detect lymphatic endothelial cells. The aim of this study was to determine the correlation between the expression of endoglin/podoplanin and clinical variables associated with OSCC progression. Materials and Methods: Paraffin embedded tissue specimens from 21 patients diagnosed with OSCC were used in this study. Ten patients were diagnosed with early clinical stage (I or II) and 11 patients with advanced clinical stage (III or IV) OSCC. Five patients had positive lymph node involvement. Primary antibodies for endoglin and podoplanin were used to perform the immunohistochemical detection of the vascular and lymphatic endothelial cells. The expression of endoglin and podoplanin was examined by an image analysis program in the three most highly expressed regions of each specimen. Results: The average endoglin expression was observed to be $1.691{\pm}0.920$ in the advanced stage (III, IV) specimens and $0.797{\pm}0.583$ in the early stage (I, II) specimens (P=0.020). The average expression of podoplanin was $0.286{\pm}0.228$ in the advance stage (III, IV) specimens and $0.374{\pm}0.157$ in the early stage (I, II) specimens (P>0.05). There was no statistically significant difference in the expression of endoglin and podoplanin, regardless of whether or not the lymph node was positive. Conclusion: The expression of endoglin was significantly higher in the advanced stage specimens than that in the early stage specimens. Therefore, we concluded that endoglin is a useful molecular marker for use in the evaluation of the progression of OSCC.

• Archives of Plastic Surgery
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• v.48 no.5
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• pp.559-567
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• 2021

The potential to differentiate into different cell lines, added to the easy and cost-effective method of extraction, makes adipose-derived stem cells (ADSCs) an object of interest in lymphedema treatment. Our study's goal was to conduct a comprehensive systematic review of the use of ADSCs in lymphatic tissue engineering and regeneration. On July 23, 2019, using PubMed/MEDLINE, Cochrane Clinical Answers, Cochrane Central Register of Controlled Trials, and Embase databases, we conducted a systematic review of published literature on the use of ADSCs in lymphatic tissue engineering and regeneration. There were no language or time frame limitations, and the following search strategy was applied: ((Adipose stem cell) OR Adipose-derived stem cell)) AND ((Lymphedema) OR Breast Cancer Lymphedema). Only original research manuscripts were included. Fourteen studies fulfilled the inclusion criteria. Eleven studies were experimental (in vitro or in vivo in animals), and only three were clinical. Publications on the topic demonstrated that ADSCs promote lymphangiogenesis, and its effect could be enhanced by modulation of vascular endothelial growth factor-C, interleukin-7, prospero homeobox protein 1, and transforming growth factor-β1. Pilot clinical studies included 11 patients with breast cancer-related lymphedema, and no significant side effects were present at 12-month follow-up. Literature on the use of ADSCs in lymphatic tissue engineering and regeneration demonstrated promising data. Clinical evidence is still in its infancy, but the scientific community agrees that ADSCs can be useful in regenerative lymphangiogenesis. Data collected in this review indicate that unprecedented advances in lymphedema treatment can be anticipated in the upcoming years.

• Korean Journal of Bronchoesophagology
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• v.8 no.1
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• pp.35-41
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• 2002

Background and Objectives : Angiogenesis within malignant tumors has been considered to be essential for the growth and expansion of cancer cells, especially for solid tumors, and has been implicated in the overall growth and metastases of tumors. Such angiogenesis within tumors depends upon the secretion of vascular growth factor to allow the growth of newly formed vessels from peripheral tissue into the malignant tumor. %n, an exploration of the relations between cancer cells and vascular growth factors is absolutely critical to understanding the growth of malignant tumors. According to recent reports, vascular endothelial growth factor(VEGF) has been found to play a role in lymphatic metastases, tumor recurrence and survival in various human tumors. To evaluate the role of VEGF in head and neck squamous cell carcinoma(HNSCC) we performed this study. Materials and Methods : We examined the expression of VEGF and microvessel density in 39 HNSCC by immunohistochemistry and correlated them with various clinical data such as stage, cervical lymphatic metastasis, recurrence, and overall survival. Results : The expression of VEGF was not correlated with overall stage, T stage and N stage. There was no statistical correlation between the expression of VEGF and recurrence in the Primary site, cervical lymph node, and the distant metastases. There was no statistical correlation between the expression of VEGF and microvessel density. Conclusion : Based on these results, it is suggested that the expression of vascular endothelial growth factor is not a major prognostic factor for head and neck squamous cell carcinoma. Further studies are needed to evaluate significance of VEGF expression in head and neck squamous cell carcinoma.

• Asian Pacific Journal of Cancer Prevention
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• v.15 no.3
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• pp.1363-1367
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• 2014

This research was conducted to compare differences in colon cancer lymphatic vessel invasion (LVI) with D2-40 antibody labeling and regular HE staining, blood vessel invasion (BVI) with CD34 antibody labeling and HE staining and to assess the possibility of using D2-40-LVI/CD34-BVI in combination for predicting stage II colon cancer prognosis and guiding adjuvant chemotherapy.Anti-D2-40 and anti-CD34 antibodies were applied to tissue samples of 220 cases of stage II colon cancer to label lymphatic vessels and small blood vessels, respectively. LVI and BVI were assessed and multivariate COX regression analysis was performed for associations with colon cancer prognosis. Regular HE staining proved unable to differentiate lymphatic vessels from blood vessels, while D2-40 selectively labeled lymphatic endothelial cell cytosol and CD34 was widely expressed in large and small blood vessels of tumors as well as normal tissues. Compared to regular HE staining, D2-40-labeling for LVI and CD34-labeling for BVI significantly increased positive rate (22.3% vs 10.0% for LVI, and 19.1% vs 9.1% for BVI). Multivariate analysis indicated that TNM stage, pathology tissue type, post-surgery adjuvant chemotherapy, D2-40-LVI, and CD34-BVI were independent factors affecting whole group colon cancer prognosis, while HE staining-BVI, HE staining-LVI were not significantly related. When CD34-BVI/D2-40-LVI were used in combination for detection, the risk of death for patients with two or one positive results was 5.003 times that in the LVI(-)&BVI(-) group (95% CI 2.365 - 9.679). D2-40 antibody LVI labeling and CD34 antibody BVI labeling have higher specificity and accuracy than regular HE staining and can be used as molecular biological indicators for prognosis prediction and guidance of adjuvant chemotherapy for stage II colon cancer.