Endogenous origin of the lymphatics in the avian chorioallantoic membrane.

The lymphatics of the intestinal organs have important functions in transporting chyle toward the jugulosubclavian junction, but the lymphangiogenic potential of the splanchnic mesoderm has not yet been tested. Therefore, we studied the allantoic bud of chick and quail embryos. It is made up of endoderm and splanchnic mesoderm and fuses with the chorion to form the chorioallantoic membrane (CAM) containing both blood vessels and lymphatics. In day 3 embryos (stage 18 of Hamburger and Hamilton [HH]), the allantoic mesoderm consists of mesenchymal cells that form blood islands during stage 19 (HH). The endothelial network of the allantoic bud, some intraluminal and some mesenchymal cells express the hemangiopoietic marker QH1. The QH1-positive endothelial cells also express the vascular endothelial growth factor receptor-3 (VEGFR-3), whereas the integrating angioblasts and the round hematopoietic cells are QH1-positive/VEGFR-3-negative. The ligand, VEGF-C, is expressed ubiquitously in the allantoic bud, and later predominantly in the allantoic epithelium and the wall of larger blood vessels. Allantoic buds of stage 17-18 (HH) quail embryos were grafted homotopically into chick embryos and reincubated until day 13. In the chimeric CAMs, quail endothelial cells are present in blood vessels and lymphatics, the latter being QH1 and VEGFR-3 double-positive. QH1-positive hematopoietic cells are found at many extra- and intraembryonic sites, whereas endothelial cells are confined to the grafting site. Our results show that the early allantoic bud contains hemangioblasts and lymphangioblasts. The latter can be identified with Prox1 antibodies and mRNA probes in the allantoic mesoderm of day 4 embryos (stage 21 HH). Prox1 is a specific marker of the lymphatic endothelium throughout CAM development.     

VEGFR-3 in adult angiogenesis.

Vascular endothelial growth factor receptor 3 (VEGFR-3, Flt-4), the receptor for vascular endothelial growth factors (VEGFs) C and D, is expressed on lymphatic endothelium and may play a role in lymphangiogenesis. In embryonic life, VEGFR-3 is essential for blood vessel development. The purpose of this study was to investigate whether VEGFR-3 is also involved in blood vessel angiogenesis in the adult. This was studied in human tissues showing angiogenesis and in a model of VEGF-A-induced iris neovascularization in the monkey eye, by the use of immunohistochemistry at the light and electron microscopic level. VEGFR-3 was expressed on endothelium of proliferating blood vessels in tumours. In granulation tissue, staining was observed in the proliferative superficial zone in plump blood vessel sprouts, in the intermediate zone in blood vessels and long lymphatic sprouts, and in the deeper fibrous zone in large lymphatics, in a pattern demonstrating that lymphangiogenesis follows behind blood vessel angiogenesis in granulation tissue formation. At the ultrastructural level, VEGFR-3 was localized in the cytoplasm and on the cell membrane of endothelial cells of sprouting blood vessels and sprouting lymphatics. In monkey eyes injected with VEGF-A, blood vessel sprouts on the anterior iris surface and pre-existing blood vessels in the iris expressed VEGFR-3. In conclusion, these results support a role for VEGFR-3 and its ligands VEGF-C and/or VEGF-D in cell-to-cell signalling in adult blood vessel angiogenesis. The expression of VEGFR-3 in VEGF-A-induced iris neovascularization and in pre-existing blood vessels exposed to VEGF-A suggests that this receptor and possibly its ligands are recruited in VEGF-A-driven angiogenesis.     

Lymphangiogenesis and lymphatic metastasis in breast cancer

Lymphatic metastasis is the main prognostic factor for survival of patients with breast cancer and other epithelial malignancies. Mounting clinical and experimental data suggest that migration of tumor cells into the lymph nodes is greatly facilitated by lymphangiogenesis, a process that generates new lymphatic vessels from pre-existing lymphatics with the aid of circulating lymphatic endothelial progenitor cells. The key protein that induces lymphangiogenesis is vascular endothelial growth factor receptor-3 (VEGFR-3), which is activated by vascular endothelial growth factor-C and -D (VEGF-C and VEGF-D). These lymphangiogenic factors are commonly expressed in malignant, tumor-infiltrating and stromal cells, creating a favorable environment for generation of new lymphatic vessels. Clinical evidence demonstrates that increased lymphatic vessel density in and around tumors is associated with lymphatic metastasis and reduced patient survival. Recent evidence shows that breast cancers induce remodeling of the local lymphatic vessels and the regional lymphatic network in the sentinel and distal lymph nodes. These changes include an increase in number and diameter of tumor-draining lymphatic vessels. Consequently, lymph flow away from the tumor is increased, which significantly increases tumor cell metastasis to draining lymph nodes and may contribute to systemic spread. Collectively, recent advances in the biology of tumor-induced lymphangiogenesis suggest that chemical inhibitors of this process may be an attractive target for inhibiting tumor metastasis and cancer-related death. Nevertheless, this is a relatively new field of study and much remains to be established before the concept of tumor-induced lymphangiogenesis is accepted as a viable anti-metastatic target. This review summarizes the current concepts related to breast cancer lymphangiogenesis and lymphatic metastasis while highlighting controversies and unanswered questions.     

Lymphangiogenic growth factor responsiveness is modulated by postnatal lymphatic vessel maturation

Lymphatic vessel plasticity and stability are of considerable importance when attempting to treat diseases associated with the lymphatic vasculature. Development of lymphatic vessels during embryogenesis is dependent on vascular endothelial growth factor (VEGF)-C but not VEGF-D. Using a recombinant adenovirus encoding a soluble form of their receptor VEGFR-3 (AdVEGFR-3-Ig), we studied lymphatic vessel dependency on VEGF-C and VEGF-D induced VEGFR-3 signaling in postnatal and adult mice. Transduction with AdVEGFR-3-Ig led to regression of lymphatic capillaries and medium-sized lymphatic vessels in mice under 2 weeks of age without affecting collecting lymphatic vessels or the blood vasculature. No effect was observed after this period. The lymphatic capillaries of neonatal mice also regressed partially in response to recombinant VEGFR-3-Ig or blocking antibodies against VEGFR-3, but not to adenovirus-encoded VEGFR-2-Ig. Despite sustained inhibitory VEGFR-3-Ig levels, lymphatic vessel regrowth was observed at 4 weeks of age. Interestingly, whereas transgenic expression of VEGF-C in the skin induced lymphatic hyperplasia even during embryogenesis, similar expression of VEGF-D resulted in lymphangiogenesis predominantly after birth. These results indicate considerable plasticity of lymphatic vessels during the early postnatal period but not thereafter, suggesting that anti-lymphangiogenic therapy can be safely applied in adults.     

B lymphocyte-specific c-Myc expression stimulates early and functional expansion of the vasculature and lymphatics during lymphomagenesis

Expression of the c-myc proto-oncogene is deregulated in many human cancers. We examined the role of c-Myc in stimulating angiogenesis and lymphangiogenesis in a highly metastatic murine model of Burkitt's lymphoma (E micro -c-myc), where c-Myc is expressed exclusively in B lymphocytes. Immunohistochemical analysis of bone marrow and lymph nodes from young (preneoplastic) E micro -c-myc transgenic mice revealed increased growth of blood vessels, which are functional by dye flow assay. Lymphatic sinuses also increased in size and number within the lymph nodes, as demonstrated by immunostaining for with a lymphatic endothelial marker 10.1.1. The 10.1.1 antibody recognizes VEGFR-2- and VEGFR-3-positive lymphatic sinuses and vessels within lymph nodes, and also recognizes lymphatic vessels in other tissues. Subcutaneously injected dye traveled more efficiently through draining lymph nodes in E micro -c-myc mice, indicating that these hypertrophic lymphatic sinuses increase lymph flow. Purified B lymphocytes and lymphoid tissues from E micro -c-myc mice expressed increased levels of vascular endothelial growth factor (VEGF) by immunohistochemical or immunoblot assays, which could promote blood and lymphatic vessel growth through interaction with VEGFR-2, which is expressed on the endothelium of both vessel types. These results indicate that constitutive c-Myc expression stimulates angiogenesis and lymphangiogenesis, which may promote the rapid growth and metastasis of c-Myc-expressing cancer cells, respectively.     

The lymphatic endothelium of the avian wing is of somitic origin.

It has recently been shown that there are lymphangioblasts in the early avian wing bud, but fate map studies on the origin of these cells have not yet been performed. The lymphatics in the wings of 10-day-old chick and quail embryos are characterized by both the position along with all major blood vascular routes and by the Vascular Endothelial Growth Factor Receptor-3 (VEGFR-3) expression. In the quail, the endothelium of both blood vessels and lymphatics can be marked with the QH1 antibody. We have grafted the dorsal halves of epithelial somites of 2-day-old quail embryos homotopically into chick embryos. The grafting was performed at the wing level and the host embryos were reincubated until day 10. The chimeric wings were studied with the QH1 antibody alone and with double staining consisting of VEGFR-3 in situ hybridization and QH1 immunofluorescence. Our results show that in the wing the endothelium of both the blood vessels and the lymphatics is derived from the somites. QH1-positive endothelial cells form the vasculature of the chimeric wings. Chimeric lymphatics of the wing can be identified because of their typical position and their VEGFR-3 and QH1 double-positivity. This shows that not only the blood vascular cells but also the lymphatic endothelial cells of the avian wing are born in the paraxial/somitic mesoderm.     

Transgenic induction of vascular endothelial growth factor-C is strongly angiogenic in mouse embryos but leads to persistent lymphatic hyperplasia in adult tissues

Vascular endothelial growth factor-C (VEGF-C) is the quintessential lymphangiogenic growth factor that is required for the development of the lymphatic system and is capable of stimulating lymphangiogenesis in adults by activating its receptor, VEGFR-3. Although VEGF-C is a major candidate molecule for the development of prolymphangiogenic therapy for defective lymphatic vessels in lymphedema, the stability of lymph vessels generated by exogenous VEGF-C administration is not currently known. We studied VEGF-C-stimulated lymphangiogenesis in inducible transgenic mouse models in which growth factor expression can be spatially and temporally controlled without side effects, such as inflammation. VEGF-C induction in adult mouse skin for 1 to 2 weeks caused robust lymphatic hyperplasia that persisted for at least 6 months. VEGF-C induced lymphangiogenesis in numerous tissues and organs when expressed in the vascular endothelium in either neonates or adult mice. Very few or no effects were observed in either blood vessels or collecting lymph vessels. Additionally, VEGF-C stimulated lymphangiogenesis in embryos after the onset of lymphatic vessel development. Strikingly, a strong angiogenic effect was observed after VEGF-C induction in vascular endothelium at any point before embryonic day 16.5. Our results indicate that blood vessels can undergo VEGF-C-induced angiogenesis even after down-regulation of VEGFR-3 in embryos; however, transient VEGF-C expression in adults can induce long-lasting lymphatic hyperplasia with no obvious side effects on the blood vasculature.     

Expression of the Vascular Endothelial Growth Factor C Receptor VEGFR-3 in Lymphatic Endothelium of the Skin and in Vascular Tumors

It is difficult to identify lymph vessels in tissue sections by histochemical staining, and thus a specific marker for lymphatic endothelial cells would be more practical in histopathological diagnostics. Here we have applied a specific antigenic marker for lymphatic endothelial cells in the human skin, the vascular endothelial growth factor receptor-3 (VEGFR-3), and show that it identifies a distinct vessel population both in fetal and adult skin, which has properties of lymphatic vessels. The expression of VEGFR-3 was studied in normal human skin by in situ hybridization, iodinated ligand binding, and immunohistochemistry. A subset of developing vessels expressed the VEGFR-3 mRNA in fetal skin as shown by in situ hybridization and radioiodinated vascular endothelial growth factor (VEGF)-C bound selectively to a subset of vessels in adult skin that had morphological characteristics of lymphatic vessels. Monoclonal antibodies against the extracellular domain of VEGFR-3 stained specifically endothelial cells of dermal lymph vessels, in contrast to PAL-E antibodies, which stained only blood vessel endothelia. In addition, staining for VEGFR-3 was strongly positive in the endothelium of cutaneous lymphangiomatosis, but staining of endothelial cells in cutaneous hemangiomas was weaker. These results establish the utility of anti-VEGFR-3 antibodies in the identification of lymphovascular channels in the skin and in the differential diagnosis of skin lesions involving lymphatic or blood vascular endothelium.     

Intratumoral Lymphatics and Lymphatic Vessel Invasion Detected by D2‐40 Are Essential for Lymph Node Metastasis in Bladder Transitional Cell Carcinoma

Bladder cancer is frequently associated with regional lymph node metastasis at the time of diagnosis or after initial treatment, and lymph node metastasis is crucial for clinical therapeutic strategies. Lymphangiogenesis, detected by antibodies specific for lymphatic endothelial cells, is correlated with cancer spread, but the mechanisms that underlie lymphatic spread and the role of lymphangiogenesis in cancer metastasis has been less clear. The aim of this study was to investigate the association of vascular endothelial growth factor (VEGF)‐D expression, intratumoral lymphatics, and lymphatic invasion associated with lymph node metastasis as well as the prognostic analysis in patients with bladder transitional cell carcinoma (TCC). The VEGF‐D expression was evaluated by immunohistochemistry in 72 specimens, and tumoral lymphatic vessels were measured by D2‐40. Counts of lymph vessels were taken in intratumoral and peritumoral areas. Survival analyses and their independent roles were investigated using univariate and multivariate analysis models. The high expression of VEGF‐D was closely associated with the intratumoral lymphatic vessels, tumoral lymphatic invasion, and lymph node metastasis as well as a shorter overall survival. Higher lymphatic vessel density, intratumoral lymphatics, and lymphatic invasion showed a significant association with lymph node metastasis. Univariate analysis indicated that VEGF‐D, intratumoral lymphatics, and lymphatic invasion were associated with overall survival, but they were not independent prognostic factors for bladder TCC in multivariate analysis. We conclude that VEGF‐D plays an essential role in tumoral lymphangiogenesis. Intratumoral lymphatics and lymphatic invasion are important predictive factors of pelvic lymph node metastasis in patients with bladder cancer. Anat Rec, 2010. © 2010 Wiley‐Liss, Inc.     

Lack of lymphangiogenesis in human primary cutaneous melanoma. Consequences for the mechanism of lymphatic dissemination.

Cutaneous melanoma has an initial preference for lymphatic spread. Remarkably, melanoma progression toward this metastasizing phenotype is accompanied by intense blood vessel angiogenesis (hemangiogenesis), but lymphangiogenesis, the formation of new lymph vessels in the tumor, has never been reported. To investigate how primary melanoma cells interact with the existing lymphatic microvasculature, and whether lymphangiogenesis occurs, an immunostaining was developed that differentially decorates blood and lymph vessels in frozen tissue sections. The density and distribution of both these vessel types in and around thin (< or = 1.5 mm) and thick (> or = 1.5 mm) primary melanoma lesions and in normal and uninvolved skin were determined. Although especially in thick melanoma lesions a significant increase in blood vessel density was observed, lymphatic density remained unaltered, showing that lymphangiogenesis did not occur. Morphological analysis indicated, however, that melanoma progression is accompanied by a sequence of events that involves hemangiogenesis supporting tumor expansion, especially in the vertical growth phase. Often, stromal sepia are formed around the blood capillaries in the tumor neovasculature protecting them from invasion. Lymph vessels inside the tumor were infrequently observed. However, subepidermal lymph vessels often seemed to be entrapped and penetrated by the expanding tumor mass. In this way, hemangiogenesis, as the driving force behind tumor expansion, might indirectly increase the chance of lymphatic invasion in the absence of lymphangiogenesis. This model explains the paradox that, although melanoma metastasis seems to require angiogenesis, a consistent relation of prognosis with blood capillary density in primary cutaneous melanoma is lacking.     

淋巴管内皮祖细胞在淋巴管新生中的作用及其机制

淋巴管新生在胚胎发育、外伤修复、炎症转归、肿瘤转移和器官移植后排斥反应等方面起着重要作用.除淋巴管内皮细胞外,淋巴管内皮祖细胞参与淋巴管新生.淋巴管内皮祖细胞表达CD34、CD133和VEGFR-3,在趋化因子作用下由骨髓动员入外周血,继而迁入局部组织.经VEGF-C等生长因子诱导向淋巴管内皮细胞分化,参与淋巴管新生....     

Vascular endothelial growth factor receptor-3 in lymphangiogenesis in wound healing

Vascular endothelial growth factor receptor-3 (VEGFR-3) is essential for embryonic cardiovascular development, but thereafter becomes confined to the lymphatic endothelium in adult tissues. We have here studied VEGFR-3 expression in experimental wounds of pigs and chronic inflammatory wounds of humans. In healing incisional and punch biopsy wounds made in the dorsal skin of pigs, angiogenic blood vessels, identified by use of the blood vascular endothelial markers vWF and PAL-E and the basal lamina protein laminin, developed into the granulation tissue stroma from day 4 onward, being most abundant on days 5 and 6 and regressing thereafter. VEGFR-3-positive vessels were observed in the granulation tissue from day 5 onward. These vessels were distinct from the PAL-E/laminin/vWF-positive vessels and fewer in number, and they appeared to sprout from pre-existing VEGFR-3-positive lymphatic vessels at the wound edge. Unlike the blood vessels, very few VEGFR-3-positive lymphatic vessels persisted on day 9 and none on day 14. In chronic wounds such as ulcers and decubitus wounds of the lower extremity of humans, VEGFR-3 was also weakly expressed in the vascular endothelium. Our results suggest that transient lymphangiogenesis occurs in parallel with angiogenesis in healing wounds and that VEGFR-3 becomes up-regulated in blood vessel endothelium in chronic inflammatory wounds.     

血管内皮生长因子D和血管内皮生长因子受体3在人结肠癌中的表达及淋巴道转移中的作用

目的 观察血管内皮生长因子D(VEGF-D)和血管内皮生长因子受体3(VEGFR-3)在人结肠癌组织中的表达,检测结肠癌组织中的微淋巴管密度(LMVD),探讨VEGF-D和VEGFR-3在淋巴管生成以及结肠癌淋巴道转移中的作用.方法 选择55例不同时期,不同分化程度的人结肠癌组织样本,应用免疫组织化学染色的方法,观察VEGF-D和VEGFR-3在人结肠癌组织中的表达,应用Podoplanin标记淋巴管,检测结肠癌组织中的淋巴管密度.结果 在55例结肠癌组织中,VEGF-D的阳性表达率为54.5%,明显高于在癌周正常组织内的表达(P＜0.05);结肠癌组织中VEGFR-3表达的阳性率为69.1%,明显高于在癌周正常组织内的表达(P＜0.01);并且VEGFR-3的表达与VEGF-D的表达具有显著相关性(P＜0.01).在结肠癌组织中,淋巴结转移阳性组,浸润深度超过肌层组,DukeC、D期的VEGF-D的表达水平和LMVD明显高于淋巴结转移阴性组,浸润深度未超过肌层组,Duke A、B期(P＜0.01),经计数淋巴管数量,癌组织中的LMVD明显高于癌周正常组织(P＜0.01),并且LMVD与VEGF-D的表达显著相关(P＜0.01).结论 结肠癌组织中VEGF-D的表达水平随着癌的浸润和转移程度的增强而增高,并且通过上调其受体VEGFR-3的表达而促进癌组织中淋巴管的生成,从而促进癌的浸润和转移.     

Localization of vascular endothelial growth factor-D in malignant melanoma suggests a role in tumour angiogenesis

Expression of angiogenic and lymphangiogenic factors by tumours may influence the route of metastatic spread. Vascular endothelial growth factor (VEGF) is a regulator of tumour angiogenesis, but studies of the inhibition of solid tumour growth by neutralizing anti-VEGF antibodies indicated that other angiogenic factors may be involved. VEGF-D may be an alternative regulator because like VEGF it is angiogenic and it activates VEGF receptor-2 (VEGFR-2), an endothelial cell receptor which is a key signalling molecule in tumour angiogenesis. This study reports the generation of monoclonal antibodies to the receptor-binding domain of VEGF-D and the use of these antibodies to localize VEGF-D in malignant melanoma. VEGF-D was detected in tumour cells and in vessels adjacent to immunopositive tumour cells, but not in vessels distant from the tumours. These findings are consistent with a model in which VEGF-D, secreted by tumour cells, activates endothelial cell receptors and thereby contributes to the regulation of tumour angiogenesis and possibly lymphangiogenesis. In addition, VEGF-D was detected in the vascular smooth muscle, but not the endothelium, of vessels in adult colon. The endothelium of these vessels was negative for VEGFR-2 and VEGFR-3. As VEGF receptors can be up-regulated on endothelium in response to vessel damage and ischaemia, these findings of a specific localization of VEGF-D in smooth muscle of the blood vessels suggest that VEGF-D produced by vascular smooth muscle could play a role in vascular repair by stimulating the proliferation of endothelial cells.     

血管内皮生长因子C及其受体3在人恶性黑色素瘤组织内的表达及意义

目的 观察人恶性黑色素瘤组织内血管内皮生长因子C(VEGF-C)及其受体3(VEGFR-3)的表达,探讨VEGF-C和VEGFR-3在恶性黑色素瘤淋巴管生成及淋巴道转移中的作用.方法 取人恶性黑色素瘤组织48例(石蜡标本30例,术后新鲜组织18例),应用免疫组织化学和RT-PCR技术,观察VEGF-C和VEGFR-3蛋白及mRNA在恶性黑色素瘤组织内的表达情况.以淋巴管内皮透明质酸受体(LYVE-1)标记淋巴管,计数恶性黑色素瘤组织淋巴管数密度.结果 VEGF-C和VEGFR-3蛋白主要表达于恶性黑色素瘤细胞胞浆内,在肿瘤周围的血管和淋巴管内皮上也可见VEGFR-3蛋白表达,VEGF-C和VEGFR-3蛋白在淋巴结转移组恶性黑色素瘤组织内的表达水平明显高于无淋巴结转移组(P<0.05).在18例新鲜恶性黑色素瘤中,淋巴结转移组VEGF-C和VEGFR-3mRNA的表达明显高于无淋巴结转移组(P<0.01).LYVE-1表达于肿瘤间质内的淋巴管内皮细胞,淋巴结转移组恶性黑色素瘤组织中的淋巴管数密度(LMVD)为9.845±2.454,无淋巴结转移组恶性黑色素瘤组织中的淋巴管数密度为6.534±2.193,淋巴结转移组恶性黑色素瘤组织内的淋巴管数密度明显高于无淋巴结转移组(P<0.01).结论恶性黑色素瘤组织内VEGF-C表达明显增高,并通过上调其受体VEGFR-3的表达促进恶性黑色素瘤组织内淋巴管的生成,从而促进恶性黑色素瘤的淋巴道转移.     

血管内皮生长因子C及其受体3在非小细胞肺癌组织中的表达及意义

目的　探讨血管内皮生长因子C(VEGF C)和受体 (VEGFR) 3在人非小细胞肺癌(NSCLC)组织中的表达及其与微血管、微淋巴管形成、淋巴转移、预后之间的关系。方法　对 76例人NSCLC及相应癌旁组织行VEGF C、VEGFR 3及CD34免疫组织化学染色链霉素抗生物素蛋白 过氧化物酶 (SP)法检测 ,进行淋巴管密度计数、微血管密度 (MVD)计数 ,并结合临床和病理资料进行分析。结果　NSCLC中 ,VEGF C的表达与肺癌分化程度负相关 (P =0 0 0 9)。VEGF C和VEGFR 3的表达水平与淋巴结转移呈正相关 (分别P =0 0 0 8,P =0 0 13) ,与淋巴浸润呈正相关 (分别P =0 0 2 7,P =0 0 2 0 )。VEGF C的表达与VEGFR 3在肺癌细胞中的表达呈正相关 (P =0 0 0 9)。VEGF C与淋巴管密度 (P =0 0 0 6 )、MVD(P =0 0 4 6 )呈正相关。淋巴管密度与淋巴结转移 (P =0 0 10 )、淋巴浸润 (P =0 0 19)、TNM分期 (P =0 0 15 )呈正相关 ,MVD与血行转移 (P <0 0 0 1)、TNM分期 (P <0 0 0 1)呈正相关。VEGF C阳性表达与生存时间、5年生存率呈负相关 (P <0 0 0 1)。结论　NSCLC中 ,VEGF C通过自分泌方式作用于受体VEGFR 3,促进肺癌组织生长 ,抑制分化。VEGF C促使肺癌内淋巴管形成 ,促进肺癌淋巴结转移。VEGF C和VEGFR 3表达增高、淋巴管密度增加