2277

FIGF

c-fos induced growth factor (vascular endothelial growth factor D);FIGF;c-fos induced growth factor (vascular endothelial growth factor D)

vascular endothelial growth factor D

Xp22.31

protein-coding

PURPOSE: expression of angiogenic and lymphangiogenic factors by tumors may influence the route of metastatic spread. The angiogenic factor vascular endothelial growth D (VEGF-D) is implicated in the development of lymphatic vessels and promotion of lymphatic metastases. The purpose of this study is to determine whether VEGF-D correlates with lymph node metastasis or prognosis. EXPERIMENTAL DESIGN: We assessed VEGF-D expression using immunohistochemistry in 105 invasive breast carcinomas with long-term follow-up. The relationship among VEGF-D expression, lymph node status, and other established clinicopathological parameters was assessed. Whether VEGF-D expression plays prognostic role in breast cancer was also investigated. RESULTS: VEGF-D expression was identified in 86 cases (81.9%). Positive VEGF-D was significantly correlated with lymph node metastasis (P = 0.0238) and high c-erbB-2 expression (P = 0.0211). Survival curves determined by the Kaplan-Meier method and univariate analysis demonstrated that positive VEGF-D was associated with both disease-free survival (P = 0.0023) and overall survival (P = 0.0222). In multivariate analysis using the Cox regression model, positive emerged as an independent indicator for disease-free survival (P = 0.0452). CONCLUSIONS: VEGF-D expression is associated with lymph node metastasis and may be a novel prognostic factor in breast cancer. VEGF-D may be useful in the treatment of breast cancer as a decision-making biomarker for aggressive treatment after operation.

Vascular endothelial growth factor (VEGF)-C and VEGF-D belong to the VEGF family, and are thought to be involved in lymphangiogenesis and angiogenesis. At present, this is the only known system that can induce lymphatic vessel growth in the body. However, the roles of VEGF-C and VEGF-D in breast cancer tissue have not been clarified. In the present study, we measured the mRNA expression of VEGF-C and VEGF-D in the breast cancer tissue of 109 patients by real-time polymerase chain reaction (RT-PCR). Between non-infiltrating breast cancer (n=6) and infiltrating breast cancer (n=103), there were no significant differences in the mRNA expression of VEGF-C and VEGF-D. In infiltrating cancer, the expression of HER2 exhibited a positive correlation to VEGF-C and VEGF-D mRNA expression (p=0.027, p=0.048). However, mRNA expression ofVEGF-C and VEGF-D did not exhibit any significant correlation to lymphatic vessel invasion or lymph node metastasis. Among patients without lymph node metastasis, the mRNA expression of VEGF-C and VEGF-D for patients with lymphatic vessel invasion was significantly higher than that for patients without lymphatic vessel invasion (p=0.001, p=0.050). The results suggest that, in breast cancer, VEGF-C and VEGF-D are involved in lymphatic vessel invasion prior to lymph node metastasis, and their expression decreases after lymph node metastasis occurs.

INTRODUCTION AND OBJECTIVES: Lymphatic metastasis is a common occurrence in human breast cancer, mechanisms remaining poorly understood. MDA-MB-468LN (468LN), a variant of the MDA-MB-468GFP (468GFP) human breast cancer cell line, produces extensive lymphatic metastasis in nude mice. 468LN cells differentially express alpha9beta1 integrin, a receptor for lymphangiogenic factors VEGF-C/-D. We explored whether (1) differential production of VEGF-C/-D by 468LN cells provides an autocrine stimulus for cellular motility by interacting with alpha9beta1 and a paracrine stimulus for lymphangiogenesis in vitro as measured with capillary-like tube formation by human lymphatic endothelial cells (HMVEC-dLy); (2) differential expression of alpha9 also promotes cellular motility/invasiveness by interacting with macrophage derived factors; (3) stable knock-down of VEGF-D or alpha9 in 468LN cells abrogates lymphangiogenesis and lymphatic metastasis in vivo in nude mice. RESULTS: A comparison of expression of cyclo-oxygenase (COX)-2 (a VEGF-C/-D inducer), VEGF-C/-D and their receptors revealed little COX-2 expression by either cells. However, 468LN cells showed differential VEGF-D and alpha9beta1 expression, VEGF-D secretion, proliferative, migratory/invasive capacities, latter functions being stimulated further with VEGF-D. The requirement of alpha9beta1 for native and VEGF-D-stimulated proliferation, migration and Erk activation was demonstrated by treating with alpha9beta1 blocking antibody or knock-down of alpha9. An autocrine role of VEGF-D in migration was shown by its impairment by silencing VEGF-D and restoration with VEGF-D. 468LN cells and their soluble products stimulated tube formation, migration/invasiveness of HMVEC-dLy cell in a VEGF-D dependent manner as indicated by the loss of stimulation by silencing VEGF-D in 468LN cells. Furthermore, 468LN cells showed alpha9-dependent stimulation of migration/invasiveness by macrophage products. Finally, capacity for intra-tumoral lymphangiogenesis and lymphatic metastasis in nude mice was completely abrogated by stable knock-down of either VEGF-D or alpha9 in 468LN cells. CONCLUSION: Differential capacity for VEGF-D production and alpha9beta1 integrin expression by 468LN cells jointly contributed to their lymphatic metastatic phenotype.

BACKGROUND: Mounting clinical and experimental data suggest that the migration of tumor cells into lymph nodes is greatly facilitated by lymphangiogenesis. Vascular endothelial growth factor (VEGF)-C and D have been identified as lymphangiogenic growth factors and play an important role in tumor lymphangiogenesis. The purpose of this study was to investigate the location of lymphangiogenesis driven by tumor-derived VEGF-C/D in breast cancer, and to determine the role of intratumoral and peritumoral lymphatic vessel density (LVD) in lymphangiogenesis in breast cancer. METHODS: The expression levels of VEGF-C/D were determined by immunohistochemistry, and intratumoral LVD and peritumoral LVD were assessed using immunohistochemistry and the D2-40 antibody in 73 patients with primary breast cancer. The associations of intratumoral LVD and peritumoral LVD with VEGF-C/D expression, clinicopathological features and prognosis were assessed. RESULTS: VEGF-C and D expression were significantly higher in breast cancer than benign disease (P < 0.01). VEGF-C (P < 0.001) and VEGF-D (P = 0.005) expression were significantly associated with peritumoral LVD, but not intratumoral LVD. Intratumoral LVD was associated with tumor size (P = 0.01). Peritumoral LVD was significantly associated with lymph node metastasis (LNM; P = 0.005), lymphatic vessel invasion (LVI; P = 0.017) and late tumor,node, metastasis (TNM) stage (P = 0.011). Moreover, peritumoral LVD was an independent risk factor for axillary lymph node metastasis, overall survival and disease-free survival in multivariate analysis. CONCLUSIONS: This study suggests that tumor-derived VEGF-C/D induce peritumoral lymphangiogenesis, which may be one mechanism that leads to lymphatic invasion and metastatic spread. Peritumoral LVD has potential as an independent prognostic factor in breast cancer patients.

BACKGROUND: To investigate the role of vascular endothelial growth factors (VEGF)-C/D and their receptor Flt-4 in the lymphatic metastasis of early-stage invasive cervical carcinoma. METHODS: Immunohistochemical (IHC) staining with the antibodies against VEGF-C, VEGF-D, and Flt-4 was used to examine the expression of them in 97 cases of early-stage cervical carcinoma (Ia-IIa). Meanwhile, the lymphatic vessel density (LVD) was measured using the antibody against lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1). We then analyzed the correlation between Flt-4-positive vessel density (FVD), LVD and clinicopathological features of the tumors. RESULTS: (1) The positive rates of VEGF-C, VEGF-D, and Flt-4 were 57.7%, 60.8%, and 52.6% in the cervical tumor samples, respectively. (2) The expression levels of VEGF-C, VEGF-D, and Flt-4 were significantly correlated with lymphatic metastasis and lymphatic vessel invasion. LVD was significantly associated with lymph node metastasis and lymphatic vessel invasion. On the other hand, FVD was strongly associated with clinical staging. (3) The expression levels of VEGF-C and VEGF-D were significantly correlated with LVD and FVD, while Flt-4 levels showed no correlation with LVD or FVD. CONCLUSION: VEGF-C/D and Flt-4 may play an important role in the process of lymphatic metastasis of early-stage invasive cervical carcinoma through paracrine and autocrine mechanisms.

OBJECTIVE: expression of vascular endothelial growth factor (VEGF)-D by tumors is associated with metastasis to lymph nodes in mice. However, there are few reports concerning the clinical significance of VEGF-D protein in human carcinoma. METHODS: After confirming production of VEGF-D by eight colorectal carcinoma cell lines, we investigated relationships between the expression of VEGF-D protein, lymph node metastasis and postoperative survival in 83 colorectal carcinoma patients. mRNA levels in cell lines were evaluated using the real-time reverse transcriptase-polymerase chain reaction, and protein was detected by Western blotting in cell lines and by immunohistochemistry in resected tissues using an antibody recognizing the processed form of the molecule. RESULTS: Immunohistochemistry showed VEGF-D-positive staining in 26 of the 83 carcinomas (31%). There was a significant relationship between the presence of VEGF-D protein and the incidence of lymph node metastasis (p < 0.01). Multivariate logistic regression analysis revealed that VEGF-D protein expression was an independent factor affecting lymph node metastasis (p < 0.01). Nonetheless, the presence or absence of VEGF-D protein had no significant impact on the survival of the patients (p = 0.15). CONCLUSION: These results suggest that the expression of VEGF-D protein could be useful in predicting the nodal status of colorectal carcinoma patients.

OBJECTIVE: Microvessel density (MVD) has been studied as a prognostic marker in human cancers. Quantification of lymphatic vessel density (LVD) is now possible by using new antibodies. expression of the lymphangiogenic growth factors, VEGF-C and VEGF-D, is associated with poorer clinicopathological outcomes in various tumours. The aim of this study was to quantify LVD and MVD in colorectal cancer, determine the relationship between LVD, MVD and clinicopathological variables and examine the relationship between LVD and tumour expression of VEGF-C and VEGF-D. METHOD: Thirty primary colorectal cancers were immunostained for CD34, lymph vessel endothelial hyaluronan receptor-1 (LYVE-1), VEGF-A and VEGF-D using standard techniques. LVD and MVD were determined by Chalkley grid counting. tumours were assessed for the presence or absence of LYVE-1 positive lymphatics at different areas within the tumour and the tumour was scored for VEGF-C and VEGF-D immunostaining intensity at the invading tumour edge. Non-parametric tests were used for statistical analysis and a P-value of <0.05 was taken as significant. RESULTS: Lymph vessel endothelial hyaluronan receptor-1 was an excellent lymphatic vessel marker. Within normal bowel wall, lymphatic vessels were found rarely in the superficial colonic mucosa, but were numerous in the submucosa and muscularis propria. In the majority of tumours, lymphatic vessels were located in the peri-tumoural area, intra-tumoural vessels were sparse and tended to be narrow with closed lumina. At the invading tumour edge, VEGF-C expression was higher (P = 0.028) and VEGF-D expression lower (P = 0.011), in tumours in which lymphatic vessels were present. No significant differences between LVD and any clinicopathological variable or route of metastasis were identified. CONCLUSION: Lymphatic vessel density and MVD can be quantified in colorectal carcinoma using immunohistochemical techniques. The balance between expression of VEGF-C and VEGF-D at the invading tumour edge may enhance lymphatic metastasis, by promoting tumour lymphangiogenesis or by activation of pre-existing lymphatic vessels. No relationship was identified between LVD and clinicopathological variables.

OBJECTIVE: To investigate the relationship between clinicopathologic characteristics and expressions of vascular endothelial growth factor-C, D (VEGF-C and VEGF-D) in esophageal squamous cancer. METHODS: The tumor tissues were collected from 62 cases of esophageal squamous carcinoma, and 20 paracancerous tissues were used as control. The expressions of VEGF-C and VEGF-D were detected by immunohistochemical method, and the count of micro lymphatic density(MLD) was measured. We analyzed their relationships with tumor clinicopathologic characteristics. RESULTS: The expression rate of VEGF-C and VEGF-D in 62 cases of esophageal squamous carcinoma were 74% and 82%. MLD in tumor border tissue was obviously higher than that in the normal paracancerous tissue (5.387 +/- 4.986 vs. 1.900 +/- 2.245) (P < 0.05). The positive lymph node metastasis and advanced clinical stage were more common in the cases with higher expression of VEGF-C and VEGF-D. There were significant associations of high MLD with high expression of VEGF-C and VEGF-D, positive lymphod node status and advanced clinical stages. expression levels of VEGF-C and VEGF-D correlated significantly with each other (r = 0.691, P < 0.001). CONCLUSION: VEGF-C and VEGF-D might promote lymphatic metastasis by inducing lymphangiogenesis especially at the edge of esophageal squamous carcinoma tissue.

Vascular endothelial growth factors C (VEGF-C) and D (VEGF-D) are important lymphangiogenic factors in human cancers. We studied the expression of VEGF-C and VEGF-D using immunohistochemistry in 73 resected esophageal cancer specimens, and correlated the results with patient clinicopathologic features and survival. High expression of VEGF-C was identified in 40 (54.7%) patients, and it correlated positively with histological grade (p=0.038), tumor stage (p=0.01), depth of tumor invasion (p=0.036) and lymph node metastasis (p=0.001). In 48 of 73 (65.7%) tumors, the VEGF-D protein was also expressed at high levels. VEGF-D immunoreactivity significantly correlated with tumor location (p=0.027), size of tumor (p=0.015), histological grade (p=0.02), depth of invasion (p=0.001) and lymph node metastasis (p=0.018). In logistic multivariate analysis, high expression of VEGF-C (OR 1.941, 95%

BACKGROUND: Vascular endothelial growth factor-D (VEGF-D), the activating ligand for vascular endothelial growth factor receptor-3 (VEGFR-3), has been identified as an important endogenic tumor-associated lymphangiogenic factors which can promote lymphatic metastasis from primary tumor. Lymphatic metastasis, which is the most common metastatic method of gastric cancer (GC), is still deemed a potentially relative indicator of distant metastasis from GC. We hypothesize that VEGF-D is associated with hepatic metastasis (HM) from GC after radical gastrectomy by promoting lymphatic metastasis and/or hematogenous metastasis. METHODS: This study included 150 GC patients who underwent radical gastrectomy (75 cases presented with HM and 75 cases presented without any recurrences). VEGF-D, VEGF-C, VEGF-A, VEGFR-2, VEGFR-3, and microvessel density (MVD) were evaluated by immunohistochemistry. Besides, clinicopathologic variables and prognosis of GC patients were analyzed statistically. RESULTS: Significant associations were seen between Lauren classification and HM, VEGF-D and HM, VEGFR-3 and HM, MVD and HM, and between nodal status and HM. VEGF-D remained an independent factor for HM from GC after radical gastrectomy. Further significant association was evaluated between high-grade VEGF-D expression and short disease-free survival of HM from GC after radical gastrectomy. CONCLUSION: VEGF-D, which can induce lymphangiogenesis specifically in many kinds of malignant diseases, is an important factor for predicting HM from GC following radical gastrectomy.

OBJECTIVE: To explore the expression of vascular endothelial growth factor (VEGF) C and D in gastric cancer and its relationship with tumor angiogenesis and lymph node metastasis. METHODS: Immunohistochemistry(SABC) and real-time PCR were used to detect the expression of VEGF-C, VEGF-D protein and mRNA in 32 gastric cancer tissues and 32 normal gastric tissues. RESULTS: The positive expression rate of VEGF-C and VEGF-D in gastric cancer tissue was significantly higher than that of normal gastric tissues (P<0.01), the expression of VEGF-C and VEGF-D in the gastric cancer group and the lymph node metastasis group were significantly different (P<0.05). The expression of VEGF-C and VEGF-D in gastric carcinoma was positively correlated with lymph node metastasis (P<0.01), the expression of VEGF-C and VEGF-D in well-differentiated carcinoma, moderately differentiated carcinoma and poorly differentiated carcinoma was statistically different (P<0.05). VEGF-C and VEGF-D expressions in gastric cancer cells were not related to the patients age, sex, and lymph node distant metastasis (P>0.05). CONCLUSION: The non-intake high expression of VEGF-C and VEGF-D in gastric cancer cells is closely related to lymph node metastasis. They serve as the important reference indicator to assess the prognosis in gastric cancer patients.

BACKGROUND: The present study was designed to evaluate the impact of the tyrosine kinase ligands VEGF-A/C/D, PDGF-A/B on tumor dissemination and survival in gastric cancer. This is the first study analyzing all these parameters in a homogeneous patient population undergoing surgery. METHODS: The expression pattern of VEGF-A/C/D and PDGF-A/B was analyzed by reverse transcriptase polymerase chain reaction (RT-PCR) in 69 samples of human gastric adenocarcinoma and correlated with tumor stage and survival. RESULTS: expression of the ligand VEGF-D significantly correlated with distant metastatic disease (P=0.00001) but not with patient survival. However, VEGF-A inversely correlated with M1 and grading, PDGF-A inversely correlated with pT and pN category. In contrast, VEGF-C and PDGF-B did not have an impact on clinicopathological parameters. CONCLUSIONS: The ligand VEGF-D, rather than the other ligands or tyrosine kinase receptors analyzed, is associated with progressive disease in gastric cancer patients undergoing surgery. The VEGF-D ligand might be a helpful marker indicating disseminated disease, and targeting VEGF-D may be a potential therapeutic strategy, although limitations imposed by the selected sample population have to be considered critically.

BACKGROUND: Antigen presenting cells, in particular dendritic cells (DCs), are critical elements in antitumor immunity induction. Some of the angiogenic factors released by tumor and stroma cells, including vascular endothelial growth factor (VEGF), are thought to affect DC function. MATERIAL/METHODS: The expression of Vascular Endothelial Growth Factor (VEGF) isoforms VEGF-A (121, 145,165, 189, 206), VEGF-C and VEGF-D were determined by immunohistochemistry, Western blotting and ELISA in 46 patients with Head and Neck Squamous Cell Carcinoma (HNSCC), 30 healthy donors, and two HNSCC tumor cell lines (PCI-1 and PCI-13). RESULTS: Increased expression of VEGF-A and VEGF-C was found in tumor tissues compared to normal epithelium (P=0.001). However, VEGF-D levels were decreased in patients with cervical nodal metastasis as compared to patients with negative lymph node status. VEGF-A plasma levels were increased in patients with lymph node metastasis (266 pg/ml) compared to patients with negative lymph node status (19.8 pg/ml). Multivariate analysis demonstrated that VEGF-A correlated with microvessel density (P=0.01), disease progression (P=0.038), a reduced number of local and peripheral mature dendritic cells (DC) (P=0.015) and an increased number of peripheral immature DCs (P=0.05). DCs incubated with tumor supernatant or VEGF-A differentiated into immature DCs and did not develop full allostimulatory activity. Allogenic T cells, when co-cultured with these immature DCs, expressed the T regulatory cell marker CD25, CTLA-4, and CD45Ro, and secreted TGF-beta, VEGF-A and IL-10. CONCLUSIONS: Taken together, our results identify VEGF-A as a multifunctional factor involved in angiogenesis, tumor progression, immunosuppression and immune tolerance.

Vascular endothelial growth factor (VEGF)-C and VEGF-D influence lymphangiogenesis through the activation of the vascular endothelial growth factor receptor (VEGFR)-3. They have been implicated in lymphatic tumor spread, which is an important prognostic factor in patients with non-small cell lung carcinoma (NSCLC). Whether or not the expression of VEGF-C, -D, and VEGFR-3 correlates with clinicopathological factors in patients with T1 lung adenocarcinoma was analysed. The tumor specimens were homogenized to determine the protein expression of VEGF-C, -D, and VEGFR-3 by enzyme-linked immunosorbent assay (ELISA). RNA fractions extracted from the tumor tissues were subjected to real-time reverse transcription-polymerase chain reaction (RT-PCR) to assess the mRNA levels of VEGF-C, -D, and VEGFR-3. The expression of VEGF-D protein and mRNA levels in patients without lymph node metastasis were significantly higher than those with metastasis (p=0.013, p=0.0494, respectively). However, the protein and mRNA levels of VEGF-C and VEGFR-3 were not significantly different in patients with or without metastasis. The 5-year survival rates of the patients with high VEGF-D levels were significantly higher than those of patients with low levels (p =0.0221). No significant difference in the survival rates was observed for VEGF-C and VEGFR-3. VEGF-D may be downregulated in NSCLC tissues in comparison to adjacent normal tissue, resulting in lymph node metastasis and poor prognosis.

BACKGROUND: Vascular endothelial growth factors (VEGF)-C and -D have been reported to be lymph-specific growth factors. In this study we analyzed the intra- and peritumoral microlymphatic vessel density (MLVD) and the expression of VEGF-C and -D in lung adenocarcinoma. METHODS: Tissue samples were obtained from 48 patients who underwent surgery for lung adenocarcinoma. The expression of VEGF-C, -D and MLVD was examined by immunohistochemistry, and the correlation with lymph node metastasis and clinicopathological features was evaluated. RESULTS: Immunohistochemical analysis revealed the presence of VEGF-C and -D immunoreactivity in 70.8 % (34/48) and 58.3 % (28/48) of the tissue samples, respectively. The group with a high expression of VEGF-C and -D in the tumor margins had a higher incidence of lymph node metastasis. MLVD in both the VEGF-C and the VEGF-D positive groups was higher than that in the negative groups, and the lymph node metastasis was likewise higher. CONCLUSION: VEGF-C and -D may play important roles in lymphangiogenesis and lymph node metastasis in lung adenocarcinoma.

Regional lymph node metastasis is one of the important indicators of cutaneous malignant melanoma. Newly formed lymphatic vessels are considered to provide a route whereby tumor cells can migrate to the lymph nodes. Both vascular endothelial growth factors (VEGF) -C and -D have been confirmed to participate in tumor lymphangiogenesis, but the prognostic significance of VEGF-C, VEGF-D, and lymphangiogenesis in cutaneous malignant melanoma remains controversial. To clarify the effects of these factors and to evaluate the relationships between lymphangiogenesis, lymph node metastasis, and prognosis in patients with malignant melanoma, the expressions of VEGF-C, VEGF-D, and their receptor (VEGFR) -3 were detected by immunohistochemistry and reverse transcriptase-polymerase chain reaction. The expressions of both VEGF-C and VEGF-D proteins were concomitantly detected in the cytoplasm of the malignant cells. VEGF-C and VEGF-D expressions were associated with VEGFR-3 expression and were significantly correlated with both peritumoral lymphangiogenesis and lymph node metastasis. The incidence of peritumoral lymphatic vessels was significantly higher in lymph node metastatic melanomas than that in nonmetastatic melanomas. Univariate and multivariate analyses indicated that VEGF-C and VEGF-D were independent prognostic factors for overall survival and disease-free survival in patients with malignant melanoma. This study suggests that both VEGF-C and VEGF-D are involved in peritumoral lymphangiogenesis and lymphatic metastasis. VEGF-C and VEGF-D expression may be clinically useful indicators for prognostic evaluation in patients with cutaneous malignant melanoma.

Endothelin receptor B (ET(B)R) is a G-protein-coupled receptor overexpressed in melanoma, blood, and lymphatic endothelial cells. Given that aberrant signal transduction can be mediated through cross talk between receptors, here, we explore the functional relationship between ET(B)R and the vascular endothelial growth factor receptor (VEGFR)-3 system and how this cross talk might influence the aggressive behavior of melanoma cells. The expression of VEGFR-3 and its ligands, VEGF-C and VEGF-D, significantly increased after activating ET(B)R by ET-1 in primary and metastatic melanoma cell lines. These effects, similarly to those induced by hypoxia, were mediated by hypoxia-inducible factor (HIF)-1alpha and HIF-2alpha. ET-1 caused the phosphorylation of VEGFR-3, which was accompanied by the activation of the downstream signaling molecules, such as MAPK and AKT. Inhibition of c-Src activity or silencing of the scaffold protein beta-arrestin-1 reduced ET-1-induced VEGFR-3 phosphorylation, demonstrating that, upon ET-1 stimulus, beta-arrestin-1 is involved with c-Src in the ET(B)R-mediated VEGFR-3 transactivation. Moreover, ET-1 in combination with VEGF-C further increased VEGFR-3, MAPK, and AKT phosphorylation and markedly promoted cell migration and vasculogenic mimicry. Dual inhibition of ET(B)R and VEGFR-3 was required for the effective inhibition of these effects, as well as for VEGFR-3 phosphorylation, demonstrating that ET(B)R cross talk with VEGFR-3 enhances cell plasticity and motility. Finally, in melanoma xenografts, ET(B)R antagonist inhibited tumor growth and the activation of the VEGF-C/VEGFR-3 axis, indicating that targeting ET(B)R may improve melanoma treatment acting directly or indirectly by impairing ET(B)R cross talk with VEGFR-3.

Studies on lymph node metastasis of soft tissue sarcomas are insufficient because of its rarity. In this study, we examined the expressions of vascular endothelial growth factor (VEGF)-C and VEGF-D in soft tissue sarcomas metastasized to lymph nodes. In addition, the effects of the two molecules on the barrier function of a lymphatic endothelial cell monolayer against sarcoma cells were analyzed. We examined 7 patients who had soft tissue sarcomas with lymph node metastases and who had undergone neither chemotherapy nor radiotherapy before lymphadenectomy. Immunohistochemistry revealed that 2 of 7 sarcomas that metastasized to lymph nodes expressed VEGF-C both in primary and metastatic lesions. On the other hand, VEGF-D expression was detected in 4 of 7 primary and 7 of 7 metastatic lesions, respectively. Interestingly, 3 cases that showed no VEGF-D expression at primary sites expressed VEGF-D in metastatic lesions. Recombinant VEGF-C at 10(-8) and VEGF-D at 10(-7)and 10(-8)g/ml significantly increased the random motility of lymphatic endothelial cells compared with controls. VEGF-D significantly increased the migration of sarcoma cells through lymphatic endothelial monolayers. The fact that VEGF-D induced the migration of fibrosarcomas through the lymphatic endothelial monolayer is the probable reason for the strong relationship between VEGF-D expression and lymph node metastasis in soft tissue sarcomas. The important propensities of this molecule for the increase of lymph node metastases are not only lymphangiogenesis but also down-regulation of the barrier function of lymphatic endothelial monolayers, which facilitates sarcoma cells entering the lymphatic circulation.

BACKGROUND & OBJECTIVE: Recently, vascular endothelial growth factor-C (VEGF-C) and -D (VEGF-D) have been identified as specific lymphangiogenic factors, and their overexpression are related to lymphatic metastasis. This study was to investigate the expression and clinical significance of VEGF-C and VEGF-D in nasopharyngeal carcinoma (NPC). METHODS: The expression of VEGF-C, VEGF-D, vascular endothelial growth factor receptor-3 (VEGFR-3), and CD34 in 66 specimens of NPC and 19 specimens of nasopharyngitis tissue were detected by SP immunohistochemistry. Lymphatic microvessel density (LMVD) and microvessel density (MVD) were calculated. RESULTS: The high expression rate of VEGF-C was significantly higher in NPC than in nasopharyngitis tissues (54.5% vs. 26.3%, P<0.05). The high expression rate of VEGF-C was significantly higher in the NPC with regional lymph node metastasis than in those without, and higher in the NPC with high T stage than in those with low T stage (P<0.05). Both univariate and multivariate logistic regression analyses showed that high expression of VEGF-C was correlated to regional lymph node metastasis (P<0.05), but not to age, sex, 5-year survival, LMVD, and MVD (P>0.05). The positive rate of VEGF-D was significantly higher in NPC tissues than in adjacent non-cancerous tissues (69.7% vs. 42.1%, P<0.05). In NPC tissues, VEGF-D expression had no correlations to age, sex, T stage, regional lymph node metastasis, LMVD, and MVD (P>0.05), but was positively related to high expression of VEGF-C. The 5-year survival rate was significantly lower in VEGF-D-positive NPC than in VEGF-D-negative NPC (50.0% vs. 85.0%, P<0.05). CONCLUSION: In NPC, high expression of VEGF-C is closely correlated to regional lymph node metastasis; positive expression of VEGF-D shows no correlation to regional lymph node metastasis, but is positively related to high expression of VEGF-C and closely correlated to 5-year survival rate.

We assessed the presence of vascular endothelial growth factor (VEGF)-C, VEGF-D and their receptor VEGFR-3 by immunohistochemistry in 59 epithelial ovarian carcinomas, 11 borderline tumours and 20 benign cystadenomas. VEGF-C and VEGF-D were generally expressed in tumour cells and also in endothelia adjacent to tumour nests which showed a strong staining for them. VEGFR-3 was expressed in lymphatic and vascular endothelial cells adjacent to tumour nests. Immunoreactivity was significantly more frequent as lesions progressed from a benign tumour to advanced carcinoma. A strong correlation was found between VEGF-C and VEGF-D detected in carcinoma and VEGFR-3 detected in neighbouring endothelial cells. Increased expression of VEGF-C, VEGF-D and VEGFR-3 was significantly associated with lymph node metastasis and peritoneal metastasis outside the pelvis. There was a significant correlation between the high levels of VEGF-C and VEGF-D proteins, and poor survival. The presence of VEGF-D was an independent prognostic indicator by multivariate analysis. We conclude that VEGF-C, VEGF-D and VEGFR-3 play an important role in lymphatic spread and intraperitoneal tumour development in ovarian carcinoma. Since VEGF-D was found to be an independent predictor of poor outcome, its measurement, together with other prognostic markers may improve prospective identification of patients with a poor prognosis.

BACKGROUND: Vascular endothelial growth factor (VEGF)-D has been shown to promote lymph node metastasis in several cancers. Although generally overexpressed in ovarian carcinoma, its role in nodal dissemination of this cancer is unclear. To clarify the role of VEGF-D and the underlying molecular mechanisms, we investigated the function of VEGF-D using a mouse xenograft model of ovarian cancer. METHODS: Human ovarian serous adenocarcinoma SKOV3 cells were transfected with VEGF-D recombinant plasmid DNA, or with control vectors. The cells were injected subcutaneously into the footpads of nude mice. tumor growth was evaluated weekly. Draining lymphatics were observed grossly with Evan's blue lymphangiography. tumoral lymphatics were delineated with both Evan's blue and LYVE-1 immunostaining. tumor metastases to lymph nodes were evaluated by H&E and CA125/CD40 staining. expression of VEGF-D in primary tumors and levels of CA125 in involved lymph nodes were examined by immunohistochemistry. tumor cell apoptosis was analyzed by Hoechst dyeing. RESULTS: Mice bearing VEGF-D overexpressing xenografts showed a significantly higher rate of lymph node metastasis and markedly greater tumor volume compared with the controls. The functional lymphatic vessels were denser and enlarged in marginal and central tumor portions. Additionally, higher CA125 expression was observed in the involved lymph nodes. Mice bearing VEGF-D overexpressing xenografts also exhibited a markedly lower apoptotic index compared with the controls. CONCLUSIONS: Our data demonstrate the important role of VEGF-D in promoting lymph node metastasis by increasing tumor lymphangiogenesis, stimulating draining lymphatic vessel formation, and enhancing tumor invasiveness. Our findings show that VEGF-D can be a promising therapeutic target for ovarian cancer.

PURPOSE: The aim of this study was to evaluate the expression of vascular endothelial growth factor (VEGF)-C and -D in pancreatic cancer and to reveal its relation to lymph node metastasis. EXPERIMENTAL DESIGN: Formalin-fixed, paraffin-embedded blocks were obtained from 58 patients with pancreatic head cancer. All of the patients underwent a curative resection. The total number of resected lymph nodes was 1,058. The expressions of VEGF-C and -D were evaluated by immunohistochemical staining. To evaluate the relation to lymph node metastasis, the expressions of VEGF-C and -D between the marginal and central portions in the tumor were compared. When >25% of the tumor cells showed distinct staining, the portion was judged as high expression. RESULTS: The two groups with high expression of VEGF-C (P = 0.015) and VEGF-D (P = 0.020) in the marginal portion had a significantly higher incidence of lymph node metastasis compared with the groups with low expression, respectively. Furthermore, the group with high expression of both VEGF-C and -D in the marginal portion had a higher incidence of lymph node metastasis compared with the group with low expression (P = 0.007). The 5-year survival rate of patients with high expression of both VEGF-C and -D in the marginal portion was significantly lower than that of patients with low expression of both VEGF-C and -D (P = 0.017). CONCLUSIONS: VEGF-C and -D expression in tumor cells in the marginal portion of the tumor significantly associated with lymphatic metastasis and prognosis in patients with pancreatic head cancer.

PURPOSE: The vascular endothelial growth factor (VEGF) family plays a critical role in tumor angiogenesis and lymphangiogenesis. We characterized, at the mRNA and protein levels, the expression of VEGF-A and VEGF-D and their cognate receptors, VEGFR-1, VEGFR-2, and VEGFR-3 in early- and advanced-stage prostate cancer specimens. EXPERIMENTAL DESIGN: The levels of VEGF-A and VEGF-D mRNA in early- and advanced-stage specimens were compared using an angiogenic gene array and were confirmed by quantitative real-time PCR. Receptor protein levels and activation status were determined by immunoblotting. Spatial expression of the proteins was evaluated using immunohistochemistry with fresh and archival tissues from benign prostatic hypertrophy specimens, early-stage prostate specimens, and advanced-stage metastatic specimens. Circulating plasma levels of these growth factors were measured using ELISAs. RESULTS: We observed that expression patterns of VEGF isotypes corresponded to the prostate cancer stage: high expression of angiogenic growth factor VEGF-A was observed in early-stage prostate specimens, whereas high expression of lymphangiogenic growth factor VEGF-D was associated with advanced-stage metastatic disease. All VEGF receptors were present at variable levels in all specimens, but their activation states varied in a stage-specific manner. VEGFR-1 and, to a limited extent, VEGFR-2 were activated in early-stage specimens, whereas VEGFR-2 and VEGFR-3 were activated in advanced-stage specimens. CONCLUSIONS: Our results suggest that lymphangiogenic markers, such as VEGF-D and VEGFR-2 and VEGFR-3, may be better than angiogenic markers as targets of therapeutic intervention in advanced-stage prostate disease.

Papillary thyroid carcinoma frequently metastasizes to regional lymph nodes, and lymph node metastasis increases the risk of local regional relapse. Recent evidence suggests that vascular endothelial growth factor-D (VEGF-D) promotes lymphangiogenesis, which in turn promotes lymphatic metastasis. Therefore, the role of VEGF-D messenger RNA transcript levels and VEGF-D immunoreactivity in lymph node metastasis in papillary thyroid carcinoma was investigated. In addition, the role of blood vascular vessel, lymph vessel, and Flt-4-positive vessel densities were studied in relation to their suspected association with lymph node metastasis, and with VEGF-D expression. VEGF-D messenger RNA transcript levels by quantitative real-time reverse transcription-polymerase chain reaction and VEGF-D immunoreactivity by immunohistochemistry in 49 papillary thyroid carcinomas were also studied. This was followed by quantitative immunohistochemical staining for CD34, podoplanin, and Flt-4. Lymph node metastasis was significantly correlated with VEGF-D messenger RNA transcript levels (P=0.027) and VEGF-D immunoreactivity (P=0.019). Increased lymph vessel density was also correlated with VEGF-D expression and lymph node metastasis. In conclusion, our findings indicate that VEGF-D expression and increased lymph vessel density may have an important role for lymph node metastasis in papillary thyroid carcinoma.

OBJECTIVE: Vascular endothelial growth factor-D (VEGF-D) has been identified as one of the lymphangiogenic growth factors involved in metastatic diffusion. The aim of this study is to evaluate the serum VEGF-D levels in patients with differentiated thyroid cancer at different conditions of disease. DESIGN AND PATIENTS: We studied prospectively the VEGF-D plasma levels in 96 subjects affected by differentiated thyroid cancer. The patients were divided into three groups according to the clinical and biochemical findings: patients with no evidence of disease (Cured), patients with pathological (>1 ng/ml) stimulated thyroglobulin (Tg) (Path-Tg/rhTSH) levels only after rhTSH and patients with elevated basal Tg levels (Path-Tg/LT4). RESULTS: The serum VEGF-D concentrations in patients of group Cured were not different from the controls, while group Path-Tg/rhTSH showed baseline serum VEGF-D levels significantly lower than group Cured and controls (P < 0.001 and P < 0.01, respectively). Moreover, the patients of group Path-Tg/LT4 showed median serum cytokine concentrations at baseline not significantly different from the patients of group Path-Tg/rhTSH. The rhTSH stimulation did not modify the difference in serum VEGF-D levels in patients of group Cured and group Path-Tg/rhTSH. CONCLUSIONS: Our data demonstrate that the VEGF-D serum levels are reduced in patients with metastases of differentiated thyroid cancer, regardless of the degree of metastatic spread. It is possible that some other molecule produced by the tumoral tissue could affect the VEGF-D physiologically produced of from different tissues, thus conducting to a decrease in the VEGF-D found in blood of patients with evidence of metastatic differentiated thyroid cancer.