Therapeutic differentiation and maturation of lymphatic vessels after lymph node dissection and transplantation

Surgery or radiation therapy of metastatic cancer often damages lymph nodes, leading to secondary lymphedema. Here we show, using a newly established mouse model, that collecting lymphatic vessels can be regenerated and fused to lymph node transplants after lymph node removal. Treatment of lymph node-excised mice with adenovirally delivered vascular endothelial growth factor-C (VEGF-C) or VEGF-D induced robust growth of the lymphatic capillaries, which gradually underwent intrinsic remodeling, differentiation and maturation into functional collecting lymphatic vessels, including the formation of uniform endothelial cell-cell junctions and intraluminal valves. The vessels also reacquired pericyte contacts, which downregulated lymphatic capillary markers during vessel maturation. Growth factor therapy improved the outcome of lymph node transplantation, including functional reconstitution of the immunological barrier against tumor metastasis. These results show that growth factor-induced maturation of lymphatic vessels is possible in adult mice and provide a basis for future therapy of lymphedema.     

Conserved signaling through vascular endothelial growth (VEGF) receptor family members in murine lymphatic endothelial cells

Lymphatic vessels guide interstitial fluid, modulate immune responses by regulating leukocyte and antigen trafficking to lymph nodes, and in a cancer setting enable tumor cells to track to regional lymph nodes. The aim of the study was to determine whether primary murine lymphatic endothelial cells (mLECs) show conserved vascular endothelial growth factor (VEGF) signaling pathways with human LECs (hLECs). LECs were successfully isolated from murine dermis and prostate. Similar to hLECs, vascular endothelial growth factor (VEGF) family ligands activated MAPK and pAkt intracellular signaling pathways in mLECs. We describe a robust protocol for isolation of mLECs which, by harnessing the power of transgenic and knockout mouse models, will be a useful tool to study how LEC phenotype contributes to alterations in lymphatic vessel formation and function.     

Signalling via vascular endothelial growth factor receptor-3 is sufficient for lymphangiogenesis in transgenic mice

Vascular endothelial growth factor receptor-3 (VEGFR-3) has an essential role in the development of embryonic blood vessels; however, after midgestation its expression becomes restricted mainly to the developing lymphatic vessels. The VEGFR-3 ligand VEGF-C stimulates lymphangiogenesis in transgenic mice and in chick chorioallantoic membrane. As VEGF-C also binds VEGFR-2, which is expressed in lymphatic endothelia, it is not clear which receptors are responsible for the lymphangiogenic effects of VEGF-C. VEGF-D, which binds to the same receptors, has been reported to induce angiogenesis, but its lymphangiogenic potential is not known. In order to define the lymphangiogenic signalling pathway we have created transgenic mice overexpressing a VEGFR-3-specific mutant of VEGF-C (VEGF-C156S) or VEGF-D in epidermal keratinocytes under the keratin 14 promoter. Both transgenes induced the growth of lymphatic vessels in the skin, whereas the blood vessel architecture was not affected. Evidence was also obtained that these growth factors act in a paracrine manner in vivo. These results demonstrate that stimulation of the VEGFR-3 signal transduction pathway is sufficient to induce specifically lymphangiogenesis in vivo.     

A genetic Xenopus laevis tadpole model to study lymphangiogenesis

Lymph vessels control fluid homeostasis, immunity and metastasis. Unraveling the molecular basis of lymphangiogenesis has been hampered by the lack of a small animal model that can be genetically manipulated. Here, we show that Xenopus tadpoles develop lymph vessels from lymphangioblasts or, through transdifferentiation, from venous endothelial cells. Lymphangiography showed that these lymph vessels drain lymph, through the lymph heart, to the venous circulation. Morpholino-mediated knockdown of the lymphangiogenic factor Prox1 caused lymph vessel defects and lymphedema by impairing lymphatic commitment. Knockdown of vascular endothelial growth factor C (VEGF-C) also induced lymph vessel defects and lymphedema, but primarily by affecting migration of lymphatic endothelial cells. Knockdown of VEGF-C also resulted in aberrant blood vessel formation in tadpoles. This tadpole model offers opportunities for the discovery of new regulators of lymphangiogenesis.     

Hepatocyte growth factor promotes lymphatic vessel formation and function

The lymphatic vascular system plays a pivotal role in mediating tissue fluid homeostasis and cancer metastasis, but the molecular mechanisms that regulate its formation and function remain poorly characterized. A comparative analysis of the gene expression of purified lymphatic endothelial cells (LEC) versus blood vascular endothelial cells (BVEC) revealed that LEC express significantly higher levels of hepatocyte growth factor receptor (HGF-R). Whereas little or no HGF-R expression was detected by lymphatic vessels of normal tissues, HGF-R was strongly expressed by regenerating lymphatic endothelium during tissue repair and by activated lymphatic vessels in inflamed skin. Treatment of cultured LEC with HGF promoted LEC proliferation, migration and tube formation. HGF-induced proliferation of LEC did not require vascular endothelial growth factor receptor-3 activation, and HGF-induced cell migration was partially mediated via integrin alpha-9. Transgenic or subcutaneous delivery of HGF promoted lymphatic vessel formation in mice, whereas systemic blockade of HGF-R inhibited lymphatic function. These results identify HGF as a novel, potent lymphangiogenesis factor, and also indicate that HGF-R might serve as a new target for inhibiting pathological lymphangiogenesis.     

Soluble VEGFR-2: an antilymphangiogenic variant of VEGF receptors

The vascular endothelial growth factor (VEGF) family of secreted proteins and their receptors are major regulators of blood vessel development (hemangiogenesis) and lymphatic vessel development (lymphangiogenesis). VEGF acts through a complex system of receptor tyrosine kinases, which can be membrane bound or soluble. New data concerning the receptor system are still emerging, thus contributing to the complexity of the system. Very recently a soluble form of VEGFR-2, termed sVEGFR-2, which is a result of alternative splicing, has been discovered. Earlier, it has been shown that a secreted/soluble form of VEGFR-1, termed sVEGFR-1, is produced by alternative splicing and exerts an antihemangiogenic effect by binding VEGF-A. The newly discovered spliced variant of sVEGFR-2 binds the lymphangiogenic growth factor VEGF-C and thus inhibits VEGF-C-induced activation of VEGFR-3, consequently inhibiting lymphatic endothelial cell proliferation. Its inactivation in murine embryos permits hyperplasia of dermal lymphatics and invasion of lymphatics into the cornea. Tumor lymphangiogenesis seems to influence the metastatic behavior of malignant cells. A correlation has been found between the downregulation of sVEGFR-2 and the malignant progression of neuroblastoma, which is characterized by lymphogenic metastases in progressed stages. Data show that lymphangiogenesis is regulated by both activators and inhibitors, and its balance is crucial in health and disease.     

Tumor lymphangiogenesis and melanoma metastasis

Malignant melanomas of the skin primarily metastasize to lymph nodes, and the detection of sentinel lymph node metastases serves as an important prognostic parameter. There is now compelling evidence that melanomas can induce lymphangiogenesis (growth of lymphatic vessels), mainly at the tumor-stroma interface, and that the level of tumor lymphangiogenesis is correlated with the incidence of sentinel lymph node metastases and with disease-free survival. Thus, tumor lymphangiogenesis can serve as a novel prognostic predictor in melanoma. Vascular endothelial growth factor (VEGF)-C, released by melanoma cells and by tumor-associated macrophages, likely represents the major lymphangiogenic factor in melanoma, although other members of the VEGF family might also be involved. The recent discovery that tumors can induce a premetastatic niche, by inducing lymphatic vessel growth in sentinel lymph nodes even before metastasis, and that lymph node lymphangiogenesis enhances metastatic spread, indicates that activated lymphatic vessels represent novel targets for the detection and/or therapy of melanoma metastases.     

Immunohistochemical investigation of lymphatic vessel formation control in mouse tooth development: lymphatic vessel-forming factors and receptors in tooth development in mice

The presence of lymphatic vessels in dental pulp has recently been controversial, and no conclusion has been reached. In this study, we investigated the control of lymphangiogenesis with dental pulp development in the mouse mandibular molar using VEGF-C, VEGF-D, and VEGFR-3 as indices of lymphatic vessel-controlling factors. In addition, to distinguish blood and lymphatic vascular epithelial cells, we performed immunohistochemical analysis using von Willebrand factor (vWF) and statistical analysis. In dental papilla in the bell-stage non-calcified period, mesenchymal cells positive for VEGF-C, VEGF-D, and VEGFR-3 increased and lumen-forming endothelial cells were noted, but vWF was negative, suggesting that these were actively forming lymphatic vessels. Positive undifferentiated mesenchymal cells, an increase in endothelial cells in dental pulp, and lumen expansion were noted early after birth. Positivity was also detected in the odontoblast layer and sheath of Hertwig after birth, suggesting that these factors also play important roles in odontoblast differentiation and maturation and periodontal ligament and tooth root formation. We embryologically clarified lymphatic vessel formation in dental pulp and a process of lymphatic vessel formation from blood vessels, suggesting involvement of the surrounding tissue, odontoblasts, and sheath of Hertwig in vessel formation.     

Genesis and pathogenesis of lymphatic vessels

The lymphatic system is generally regarded as supplementary to the blood vascular system, in that it transports interstitial fluid, macromolecules, and immune cells back into the blood. However, in insects, the open hemolymphatic (or lymphohematic) system ensures the circulation of immune cells and interstitial fluid through the body. The Drosophila homolog of the mammalian vascular endothelial growth factor receptor (VEGFR) gene family is expressed in hemocytes, suggesting a close relationship to the endothelium that develops later in phylogeny. Lymph hearts are typical organs for the propulsion of lymph in lower vertebrates and are still transiently present in birds. The lymphatic endothelial marker VEGFR-3 is transiently expressed in embryonic blood vessels and is crucial for their development. We therefore regard the question of whether the blood vascular system or the lymphatic system is primary or secondary as open. Future molecular comparisons should be performed without any bias based on the current prevalence of the blood vascular system over the lymphatic system. Here, we give an overview of the structure, function, and development of the lymphatics, with special emphasis on the recently discovered lymphangiogenic growth factors.     

Lymphatic function is regulated by a coordinated expression of lymphangiogenic and anti-lymphangiogenic cytokines

Lymphangiogenic cytokines such as vascular endothelial growth factor-C (VEGF-C) are critically required for lymphatic regeneration; however, in some circumstances, lymphatic function is impaired despite normal or elevated levels of these cytokines. The recent identification of anti-lymphangiogenic molecules such as interferon-γ (IFN-γ), transforming growth factor-β1, and endostatin has led us to hypothesize that impaired lymphatic function may represent a dysregulated balance in the expression of pro/anti-lymphangiogenic stimuli. We observed that nude mice have significantly improved lymphatic function compared with wild-type mice in a tail model of lymphedema. We show that gradients of lymphatic fluid stasis regulate the expression of lymphangiogenic cytokines (VEGF-A, VEGF-C, and hepatocyte growth factor) and that paradoxically the expression of these molecules is increased in wild-type mice. More importantly, we show that as a consequence of T-cell-mediated inflammation, these same gradients also regulate expression patterns of anti-lymphangiogenic molecules corresponding temporally and spatially with impaired lymphatic function in wild-type mice. We show that neutralization of IFN-γ significantly increases inflammatory lymph node lymphangiogenesis independently of changes in VEGF-A or VEGF-C expression, suggesting that alterations in the balance of pro- and anti-lymphangiogenic cytokine expression can regulate lymphatic vessel formation. In conclusion, we show that gradients of lymphatic fluid stasis regulate not only the expression of pro-lymphangiogenic cytokines but also potent suppressors of lymphangiogenesis as a consequence of T-cell inflammation and that modulation of the balance between these stimuli can regulate lymphatic function.     

Lymphangiogenesis and breast cancer metastasis

Breast cancer is one of the commonest malignancies in women in the western world. It spreads predominantly via the lymphatic system. However, the understanding of the formation of lymphatics, lymphangiogenesis, has been limited. This has been largely due to the previous lack of lymphatic specific markers. The most specific marker used in humans has been the vascular endothelial growth factor receptor 3 (VEGFR-3). However, this is also found on blood vessel endothelium. The other vascular endothelial factor receptors (VEGFR-1 and -2) are primarily blood vessel receptors. More recently, novel, specific markers for lymphatics have been discovered, such as LYVE-1, prox I and podoplanin, enabling further research into this new field. Each of these new markers is described in detail. The article also outlines the current understanding in breast cancer metastasis, with an emphasis on the more recent research into lymphangiogenesis. Since these specific markers are now available, quantitation of lymphangiogenesis is now possible by using either immunohistochemistry or quantitative PCR approaches. In addition, to breast cancer, research into many other cancers is now possible using these methods and new markers. With this in mind, possible therapeutic strategies for the future are discussed.     

Prognostic Significance of Peritumoral Lymphatic Vessel Density and Vascular Endothelial Growth Factor Receptor 3 in Invasive Squamous Cell Cervical Cancer

Cervical cancer is known to metastasize primarily by the lymphatic system. Dissemination through lymphatic vessels represents an early step in regional tumor progression, and the presence of lymphatic metastasis is associated with a poor prognosis. In patients who have undergone a radical hysterectomy, lymphovascular space invasion (LVSI), assessed on hematoxylin and eosin-stained slides, is a major factor for adjuvant therapy in patients with cervical cancer. With the advent of a lymphatic endothelial cell-specific marker, such as D2-40, it is now possible to distinguish between blood and lymphatic space invasion (LSI). In this study, the utility of D2-40 was assessed for the detection of lymphatic vessel density (LVD) and identification of LSI. The expressions of vascular endothelial growth factor receptor-3 (VEGFR-3), VEGF-C, tyrosine receptor kinase-2, and angiopoietin-1 were assessed by immunohistochemical methods on 50 patients with squamous cell carcinoma of the cervix. Clinicopathologic characteristics, including pelvic lymph node metastasis, were correlated with the above histochemical findings. We found that lymphangiogenesis, measured by an increase in peritumoral LVD, was significantly associated with positive lymph node status (P < .005). VEGFR-3 expression was significantly associated with LVD (P < .05). D2-40 staining verified LSI (P = .03) and surpassed that of hematoxylin and eosin-identified LVSI (P = .54). In conclusion, lymphangiogenic markers, specifically LVD quantified by D2-40 and VEGFR-3, are independently associated with LSI and lymph node metastasis in patients with early squamous cell carcinoma of the cervix treated with radical hysterectomy and pelvic lymphadenectomy.     

VEGF-D的表达与膀胱移行细胞癌LVD及淋巴结转移有关

目的观察血管内皮生长因子D(vascular endothelial growth factor D,VEGF-D)在人膀胱移行细胞癌组织内的表达,探讨VEGF-D在膀胱移行细胞癌组织淋巴管密度(lymphatic vessel density,LVD)及淋巴结转移之间的关系。方法取人膀胱移行细胞癌组织蜡块30例,免疫组化法观察VEGF-D在膀胱移行细胞癌组织内的表达情况。以淋巴管内皮特异性标记物D2-40标记淋巴管,计数癌组织内淋巴管密度。结果VEGF-D蛋白主要表达于癌细胞胞浆内,VEGF-D在淋巴结转移组膀胱移行细胞癌组织内的表达水平明显高于无淋巴结转移组(P0.05);淋巴结转移组膀胱移行细胞癌组织内的淋巴管密度明显高于无淋巴结转移组(P0.05)。VEGF-D表达与膀胱移行细胞癌淋巴管密度及淋巴结转移之间具有显著的相关性。结论VEGF-D表达在膀胱移行细胞癌组织内淋巴管生成及淋巴结转移中起重要作用。     

Abnormal lymphatic vessel development in neuropilin 2 mutant mice

Neuropilin 2 is a receptor for class III semaphorins and for certain members of the vascular endothelial growth factor family. Targeted inactivation of the neuropilin 2 gene (Nrp2) has previously shown its role in neural development. We report that neuropilin 2 expression in the vascular system is restricted to veins and lymphatic vessels. Homozygous Nrp2 mutants show absence or severe reduction of small lymphatic vessels and capillaries during development. This correlated with a reduction of DNA synthesis in the lymphatic endothelial cells of the mutants. Arteries, veins and larger, collecting lymphatic vessels developed normally, suggesting that neuropilin 2 is selectively required for the formation of small lymphatic vessels and capillaries.     

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 .     

VEGF-D promotes the metastatic spread of tumor cells via the lymphatics

Metastasis to local lymph nodes via the lymphatic vessels is a common step in the spread of solid tumors. To investigate the molecular mechanisms underlying the spread of cancer by the lymphatics, we examined the ability of vascular endothelial growth factor (VEGF)-D, a ligand for the lymphatic growth factor receptor VEGFR-3/Flt-4, to induce formation of lymphatics in a mouse tumor model. Staining with markers specific for lymphatic endothelium demonstrated that VEGF-D induced the formation of lymphatics within tumors. Moreover, expression of VEGF-D in tumor cells led to spread of the tumor to lymph nodes, whereas expression of VEGF, an angiogenic growth factor which activates VEGFR-2 but not VEGFR-3, did not. VEGF-D also promoted tumor angiogenesis and growth. Lymphatic spread induced by VEGF-D could be blocked with an antibody specific for VEGF-D. This study demonstrates that lymphatics can be established in solid tumors and implicates VEGF family members in determining the route of metastatic spread.     

Bone Marrow-Derived Mesenchymal Stem Cells Drive Lymphangiogenesis

It is now well accepted that multipotent Bone-Marrow Mesenchymal Stem Cells (BM-MSC) contribute to cancer progression through several mechanisms including angiogenesis. However, their involvement during the lymphangiogenic process is poorly described. Using BM-MSC isolated from mice of two different backgrounds, we demonstrate a paracrine lymphangiogenic action of BM-MSC both in vivo and in vitro. Co-injection of BM-MSC and tumor cells in mice increased the in vivo tumor growth and intratumoral lymphatic vessel density. In addition, BM-MSC or their conditioned medium stimulated the recruitment of lymphatic vessels in vivo in an ear sponge assay, and ex vivo in the lymphatic ring assay (LRA). In vitro, MSC conditioned medium also increased the proliferation rate and the migration of both primary lymphatic endothelial cells (LEC) and an immortalized lymphatic endothelial cell line. Mechanistically, these pro-lymphangiogenic effects relied on the secretion of Vascular Endothelial Growth Factor (VEGF)-A by BM-MSC that activates VEGF Receptor (VEGFR)-2 pathway on LEC. Indeed, the trapping of VEGF-A in MSC conditioned medium by soluble VEGF Receptors (sVEGFR)-1, -2 or the inhibition of VEGFR-2 activity by a specific inhibitor (ZM 323881) both decreased LEC proliferation, migration and the phosphorylation of their main downstream target ERK1/2. This study provides direct unprecedented evidence for a paracrine lymphangiogenic action of BM-MSC via the production of VEGF-A which acts on LEC VEGFR-2.     

Heterogeneity in immunohistochemical, genomic, and biological properties of human lymphatic endothelial cells between initial and collecting lymph vessels

The immunohistochemical properties of selective lymph vessel markers, and NO synthase (NOS) and cyclo-oxygenase (COX) activities, were examined in two kinds of human lymphatic endothelial cells isolated from collecting (macro-) and initial (micro-) lymph vessels. The constitutively expressed genes in the two kinds of lymphatic endothelial cells were also evaluated by using oligonucleotide microarray analysis and RT-PCR. We also investigated the effects of oxygen concentration in culture conditions or growth factors such as basic fibroblast growth factor (bFGF), VEGF-A, and VEGF-C on proliferation activities of the two kinds of human lymphatic endothelial cells. Immunoreactivity to LYVE-1 and the RT-PCR expression level of LYVE-1 mRNA in endothelial cells of micro-lymph vessels were stronger than those of macro-lymph vessels. Immunoreactivity to VEGF R1 was also observed as significantly stronger in the micro-lymph vessels. In contrast, the immunoreactivity to Prox-1 and the RT-PCR expression level of Prox-1 mRNA in endothelial cells of macro-lymph vessels were stronger than those of micro-lymph vessels. Similarly, immunoreactivity to ecNOS, iNOS, COX1, and COX2 was also found as significantly higher than in macro-lymph vessels. In contrast, the increase of O(2) concentration ranging from 5% to 21% caused a significant reduction of the proliferation activity of endothelial cells in macro-lymph vessels. In conclusion, these findings suggest marked heterogeneity in the immunohistochemical, genomic, and proliferation activity of human lymphatic endothelial cells between micro-(initial) and macro-(collecting) lymph vessels.     

TGFBIp mediates lymphatic sprouting in corneal lymphangiogenesis

Corneal lymphangiogenesis plays a key role in diverse pathological conditions of the eye. Here, we demonstrate that a versatile extracellular matrix protein, transforming growth factor‐β induced protein (TGFBIp), promotes lymphatic sprouting in corneal lymphangiogenesis. TGFBIp is highly up‐regulated in inflamed mouse corneas. Immunolocalization of TGFBIp is detected in infiltrating macrophages in inflamed mouse corneas. Subconjunctival injection of liposomal clodronate can significantly reduce macrophage infiltration in inflamed mouse cornea, and decrease the expression of TGFBIp and areas of corneal lymphangiogenesis and angiogenesis after corneal suture placement. In brief, these results indicate that the up‐regulation of TGFBIp in sutured cornea correlates with macrophage infiltration. Although TGFBIp alone cannot significantly stimulate corneal lymph vessel ingrowth in vivo, it can enhance the effect of vascular endothelial growth factor‐C in promoting corneal lymphangiogenesis. The in vitro results show that TGFBIp promotes migration, tube formation and adhesion of human lymphatic endothelial cells (HLECs), but it has no effect on HLECs' proliferation. We also find that the in vitro effect of TGFBIp is mediated by the integrin α5β1‐FAK pathway. Additionally, integrin α5β1 blockade can significantly inhibit lymphatic sprouting induced by TGFBIp. Taken together, these findings reveal a new molecular mechanism of lymphangiogenesis in which the TGFBIp‐integrin pathways plays a pivotal role in lymphatic sprouting.