Vascular Endothelial Growth factor-C mRNA Levels Research Articles

RIP1 regulates TNF-α-mediated lymphangiogenesis and lymphatic metastasis in gallbladder cancer by modulating the NF-κB-VEGF-C pathway.

BackgroundTumor necrosis factor alpha (TNF-α) enhances lymphangiogenesis in gallbladder carcinoma (GBC) via activation of nuclear factor (NF-κB)-dependent vascular endothelial growth factor-C (VEGF-C). Receptor-interacting protein 1 (RIP1) is a multifunctional protein in the TNF-α signaling pathway and is highly expressed in GBC. However, whether RIP1 participates in the signaling pathway of TNF-α-mediated VEGF-C expression that enhances lymphangiogenesis in GBC remains unclear.MethodsThe RIP1 protein levels in the GBC-SD and NOZ cells upon stimulation with increasing concentrations of TNF-α as indicated was examined using Western blot. Lentiviral RIP1 shRNA and siIκBα were constructed and transduced respectively them into NOZ and GBC-SD cells, and then PcDNA3.1-RIP1 vectors was transduced into siRIP1 cell lines to reverse RIP1 expression. The protein expression of RIP1, inhibitor of NF-κB alpha (IκBα), p-IκBα, TAK1, NF-κB essential modulator were examined through immunoblotting or immunoprecipitation. Moreover, VEGF-C mRNA levels were measured by quantitative real-time polymerase chain reaction, VEGF-C protein levels were measured by immunoblotting and enzyme-linked immunosorbent assay, and VEGF-C promoter and NF-κB activities were quantified using a dual luciferase reporter assay. The association of NF-κB with the VEGF-C promoter was analysed by chromatin immunoprecipitation assay. A three-dimensional coculture method and orthotopic transplantation nude mice model were used to evaluate lymphatic tube-forming and metastasis ability in GBC cells. The expression of RIP1 protein, TNF-α protein and lymphatic vessels in human GBC tissues was examined by immunohistochemistry, and the dependence between RIP1 protein with TNF-α protein and lymphatic vessel density was analysed.ResultsTNF-α dose- and time-dependently increased RIP1 protein expression in the GBC-SD and NOZ cells of GBC, and the strongest effect was observed with a concentration of 50 ng/ml. RIP1 is fundamental for TNF-α-mediated NF-κB activation in GBC cells and can regulate TNF-α-mediated VEGF-C expression at the protein and transcriptional levels through the NF-κB pathway. RIP1 can regulate TNF-α-mediated lymphatic tube formation and metastasis in GBC cells both in vitro and vivo. The average optical density of RIP1 was linearly related to that of TNF-α protein and the lymphatic vessel density in GBC tissues.ConclusionWe conclude that RIP1 regulates TNF-α-mediated lymphangiogenesis and lymph node metastasis in GBC by modulating the NF-κB-VEGF-C pathway.

Interleukin-6 Induces Vascular Endothelial Growth Factor-C Expression via Src-FAK-STAT3 Signaling in Lymphatic Endothelial Cells.

Elevated serum interleukin-6 (IL-6) levels correlates with tumor grade and poor prognosis in cancer patients. IL-6 has been shown to promote tumor lymphangiogenesis through vascular endothelial growth factor-C (VEGF-C) induction in tumor cells. We recently showed that IL-6 also induced VEGF-C expression in lymphatic endothelial cells (LECs). However, the signaling mechanisms involved in IL-6-induces VEGF-C induction in LECs remain incompletely understood. In this study, we explored the causal role of focal adhesion kinase (FAK) in inducing VEGF-C expression in IL-6-stimulated murine LECs (SV-LECs). FAK signaling blockade by NSC 667249 (a FAK inhibitor) attenuated IL-6-induced VEGF-C expression and VEGF-C promoter-luciferase activities. IL-6’s enhancing effects of increasing FAK, ERK1/2, p38MAPK, C/EBPβ, p65 and STAT3 phosphorylation as well as C/EBPβ-, κB- and STAT3-luciferase activities were reduced in the presence of NSC 667249. STAT3 knockdown by STAT3 siRNA abrogated IL-6’s actions in elevating VEGF-C mRNA and protein levels. Moreover, Src-FAK signaling blockade reduced IL-6’s enhancing effects of increasing STAT3 binding to the VEGF-C promoter region, cell migration and endothelial tube formation of SV-LECs. Together these results suggest that IL-6 increases VEGF-C induction and lymphangiogenesis may involve, at least in part, Src-FAK-STAT3 cascade in LECs.

Elevated Expression of VEGF-C and Its Receptors, VEGFR-2 and VEGFR-3, in Patients with Mesial Temporal Lobe Epilepsy.

Mesial temporal lobe epilepsy (MTLE) is a frequent form of focal intractable epilepsy in adults. We previously reported overexpression of vascular endothelial growth factor C (VEGF-C) and its receptors, VEGFR-2 and VEGFR-3, in epilepsy-associated tuberous sclerosis complex. To identify whether VEGF-C and its receptors are involved in epileptogenesis of MTLE, we investigated the levels and expression pattern of VEGF-C and its receptors in temporal neocortex and hippocampus (HPC) from 28 patients with MTLE and ten control (CTX) subjects. Real-time quantitative polymerase chain reaction and Western blotting results revealed upregulated mRNA and immunoreactive protein levels of VEGF-C, VEGFR-2, and VEGFR-3 in the MTLE group compared to the control groups. Immunohistochemistry and double-labeled immunofluorescence showed that VEGF-C was highly expressed in neurons and astrocytes, including reactive astrocytes and vascular endothelial cells, VEGFR-2 was expressed at a high level in reactive astrocytes and vascular endothelial cells, but not in neurons, whereas VEGFR-3 was only overexpressed in reactive astrocytes. Taken together, these findings suggest that VEGF-C and its receptors, VEGFR-2 and VEGFR-3, may contribute to the epileptogenesis of MTLE.

Heparanase regulates in vitro VEGF-C expression and its clinical significance to pancreatic ductal cell adenocarcinoma.

Heparanase (HPSE) and vascular endothelial growth factor C (VEGF-C) are important cytokines that promote metastasis and angiogenesis in numerous malignant neoplasms, however, their association remains unclear in pancreatic ductal cell adenocarcinoma (PDAC). The present study aimed to investigate whether HPSE has a positive correlation with VEGF-C expression and to uncover the role it plays in the in vitro invasion of BxPC-3 cells (a pancreatic carcinoma cell line), and to analyze the value of joint detection of HPSE and VEGF-C for PDAC patients. A recombinant plasmid, GV230/HPSE was constructed and BxPC-3 cells were transiently transfected with GV230/HPSE or siRNA against HPSE. The expression levels of HPSE and VEGF-C were compared using reverse transcription quantitative PCR (RT-qPCR) and immunoblotting. The metastatic potential of treated BxPC-3 cells was evaluated using a Transwell® invasion assay. The relative mRNA levels of HPSE and VEGF-C in 34 PDAC specimens were assessed by RT-qPCR. The results of the RT-qPCR demonstrated a 10.7- and 3.24-fold elevation (P<0.01) of HPSE mRNA and VEGF-C mRNA, respectively, in GV230/HPSE group, whereas the HPSE siRNA group were downregulated for these mRNAs (−2.45-fold, P<0.01; −1.84-fold, P<0.01). The same pattern for protein expression was detected using immunoblot assays. In Transwell® invasion assays 138±5 cells in GV230/HPSE group and 53±4 cells in siRNA group migrated through the Matrigel®. A negative correlation between the mRNA levels of HPSE and VEGF-C in PDAC specimens and the prognosis factors of the postoperative patients was identified. Spearman rank correlation analysis indicated a positive correlation between HPSE and VEGF-C in PDAC (r=0.812, P<0.01). HPSE regulates the expression of VEGF-C and facilitates invasion of BxPC-3 in vitro. Joint detection of HPSE and VEGF-C may therefore be clinically useful in determining the prognosis of pancreatic cancer patients.

Fenofibrate inhibits the expression of VEGFC and VEGFR-3 in retinal pigmental epithelial cells exposed to hypoxia.

The aim of the present study was to examine the mechanisms through which fenofibrate inhibits the ability of human retinal pigment epithelial cells (RPE cells) exposed to hypoxia to stimulate the proliferation and migration of human umbilical vein endothelial cells (HUVECs). For this purpose, RPE cells and HUVECs were divided into the following groups: RPE-normoxia, RPE + fenofibrate, RPE-hypoxia, RPE hypoxia + fenofibrate; HUVECs normal culture and HUVECs + RPE-hypoxia culture supernatant. RPE cell hypoxia was induced by cobalt(II) chloride (CoCl2). A superoxide anion probe was used to measure the production of superoxide anion, which is indicative of hypoxic conditions. Cell proliferation was assessed by MTT assay, and the expression of vascular endothelial growth factor C (VEGFC) and vascular endothelial growth factor receptor-3 (VEGFR-3) in the RPE cell culture supernatant was measured by enzyme-linked immunosorbent assay (ELISA). The migration ability of the HUVECs was determined by scratch-wound assay, and the angiogenic ability of the HUVECs was examined by measuring cell lumen formation. The mRNA and protein expression levels of VEGFC and VEGFR-3 in the RPE cells were measured by RT-qPCR and western blot analysis, respectively. Our results revealed that fenofibrate inhibited the increase in the expression and release of VEGFC and VEGFR-3 into the RPE cell culture supernatant induced by exposure to hypoxia. The culture of HUVECs in medium supernatant of RPE cells epxosed to hypoxia enhanced the viability and migration ability of the HUVECs and promoted lumen formation; these effects were inhibited by fenofibrate. In conclusion, our data demonstrated that the exposure of RPE cells to hypoxia induced the expression and release of VEGFC and VEGFR-3 into the cell culture supernatant. The culture of HUVECs in conditioned medium from RPE cells exposed to hypoxia increased VEGFC and VEGFR-3 expression, and promoted the proliferation and migration of the HUVECs, as well as capillary tube formation, suggesting that RPE cells play an important role in the formation of choroidal neovascularization resulting from hypoxia. Fenofibrate inhibited the upregulation of VEGFC and VEGFR-3 in the RPE cells exposed to hypoxia, and thus reduced the ability of HUVECs to form new blood vessels.

Src contributes to IL6-induced vascular endothelial growth factor-C expression in lymphatic endothelial cells

Formation of lymphatic capillaries by lymphatic endothelial cells (LECs) occurs both in normal tissues as well as in pathological processes including tumor metastasis. Interleukin-6 (IL-6), a potent pro-inflammatory cytokine, has been shown to be highly elevated in various cancers. IL-6 has also been shown to increase tumor lymphangiogenesis through vascular endothelial growth factor-C (VEGF-C) induction in tumor cells. Although lymphangiogenesis is associated with lymph node metastasis and also resistance to conventional therapy in various cancers, the precise mechanisms of lymphangiogenesis in LECs remain unclear. This study aimed to investigate the signaling cascade involved in IL-6-induced VEGF-C expression in murine LECs (SV-LEC). The VEGF-C mRNA and protein levels were increased in SV-LECs exposed to IL-6. IL-6 time-dependently induced Src phosphorylation and downstream phosphorylation of ERK1/2 and p38MAPK. In contrast, PP2, an inhibitor of Src signaling, abrogated IL-6's effects on ERK1/2 and p38MAPK phosphorylation. IL-6 exposure also led to increase in VEGF-C promoter-luciferase activity as well as C/EBPβ- and κB-luciferase activities. VEGF-C promoter-, C/EBPβ- and κB-luciferase activities were all suppressed by Src, ERK1/2 or p38MAPK signaling blockades despite presence of IL-6. Finally, C/EBPβ and p65 binding to the VEGF-C promoter region were increased after IL-6 exposure in SV-LECs. Taken together, we report a Src-mediated ERK1/2 and p38MAPK activation resulting in C/EBPβ and p65 binding to the promoter region of VEGF-C, leading to VEGF-C expression in IL-6-exposed SV-LECs.

Expression and clinical significance of extracellular matrix protein 1 and vascular endothelial growth factor-C in lymphatic metastasis of human breast cancer.

BackgroundExtracellular matrix protein 1 (ECM1) and vascular endothelial growth factor-C (VEGF-C) are secretory glycoproteins that are associated with lymphangiogenesis; these proteins could, therefore, play important roles in the lymphatic dissemination of tumors. However, very little is known about their potential roles in lymphangiogenesis. The aim of this study was to investigate whether correlations exist between ECM1 and VEGF-C in human breast cancer, lymphangiogenesis, and the clinicopathological characteristics of the disease.MethodsECM1 and VEGF-C mRNA and protein expression levels in 41 patients were investigated using real-time reverse transcriptase polymerase chain reaction (RT-PCR), or immunohistochemical (IHC) staining of breast cancer tissue, matched noncancerous breast epithelial tissues, and suspicious metastatic axillary lymph nodes. D2-40 labelled lymph vessels and lymphatic microvessel density (LMVD) were counted. Correlations between ECM1 or VEGF-C protein expression levels, LMVD, and clinicopathological parameters were statistically tested.ResultsThe rate of ECM1 positive staining in breast cancer tissues was higher (31/41, 75.6%) than that in the corresponding epithelial tissues (4/41, 9.8%, P < 0.001) and lymph nodes (13/41, 31.7%, P < 0.001). Similarly, the VEGF-C expression rate in cancer specimens was higher (33/41, 80.5%) than in epithelial tissues (19/41, 46.3%, P < 0.01) or lymph nodes (15/41, 36.6%, P < 0.01). Higher ECM1 and VEGF-C mRNA expression levels were also detected in the tumor tissues, compared to the non-cancerous tissue types or lymph nodes (P < 0.05). ECM1 protein expression was positively correlated with the estrogen receptor status (P < 0.05) and LMVD (P < 0.05). LMVD in the ECM1- and VEGF-C-positive tumor specimens was higher than that in the tissue types with negative staining (P < 0.05).ConclusionsBoth ECM1 and VEGF-C were overexpressed in breast cancer tissue samples. ECM1 expression was positively correlated with estrogen responsiveness and the metastatic properties of breast cancer. We conclude, therefore, that ECM1 and VEGF-C may have a synergistic effect on lymphangiogenesis to facilitate lymphatic metastasis of breast cancer.

VEGF-C promotes the development of esophageal cancer via regulating CNTN-1 expression

Vascular endothelial growth factor C (VEGF-C) is a key regulator of angiogenesis and lymphangiogenesis. VEGF-C is also implicated in the development of esophageal cancer. We investigated the mRNA levels of VEGF-C and its receptors in 38 esophageal squamous cell carcinoma specimens (ESCCs) and matched adjacent normal esophageal tissues via real-time PCR. The mRNA levels of VEGF-C, VEGFR-2 and VEGFR-3 were significantly upregulated in ESCCs versus respective side normal tissues. To explore the influence of VEGF-C on esophageal cancer progression, the expression of VEGF-C was manipulated in esophageal cancer cell lines TE-1 and Eca-109. VEGF-C transcription, translation and secretion were significantly enhanced in cells stably transfected with a VEGF-C overexpression vector or attenuated in VEGF-C shRNA-transfected cell lines. In vitro, TE-1 cells stably transfected with a VEGF-C overexpression vector exhibited an increased rate of cell proliferation, migration and focus formation, whereas knockdown of VEGF-C inhibited cell proliferation, migration and focus formation. Similar results were obtained for Eca-109 cells. VEGF-C mediated biological function through transcription of CNTN-1, which is implicated in tumor invasion and metastasis. The expression of VEGF-C was correlated with that of CNTN-1 and cell proliferation and migration induced by VEGF-C were reversed by silencing of CNTN-1. In addition, nude mice inoculated with VEGF-C shRNA-transfected cells exhibited a significantly decreased tumor size in vivo via reduced VEGFR-2 and VEGFR-3 phosphorylation and microvessel formation. VEGF-C upregulation may be involved in esophageal tumor progression. Vector-based RNA interference (RNAi) targeting VEGF-C is a potential therapeutic method for human esophageal carcinoma.

Metastasis-associated protein 1 induces VEGF-C and facilitates lymphangiogenesis in colorectal cancer

To study the correlation between high metastasis-associated protein 1 (MTA1) expression and lymphangiogenesis in colorectal cancer (CRC) and its role in production of vascular endothelial growth factor-C(VEGF-C). Impact of high MTA1 and VEGF-C expression levels on disease progression and lymphovascular density (LVD, D2-40-immunolabeled) in 81 cases of human CRC was evaluated by immunohistochemistry. VEGF-C mRNA and protein expressions in human LoVo and HCT116 cell lines were detected by real-time polymerase chain reaction and Western blotting, respectively, with a stable expression vector or siRNA. The elevated MTA1 and VEGF-C expression levels were correlated with lymph node metastasis and Dukes stages (P < 0.05). Additionally, high MTA1 expression level was correlated with a large tumor size (P < 0.05). A significant correlation was found between MTA1 and VEGF-C protein expressions in tumor cells (r = 0.371, P < 0.05). Similar to the VEGF-C expression level, high MTA1 expression level was correlated with high LVD in CRC (P < 0.05). Furthermore, over-expression of MTA1 significantly enhanced the VEGF-C mRNA and protein expression levels, whereas siRNAs - knocked down MTA1 decreased the VEGF-C expression level. MTA1, as a regulator of tumor-associated lymphangiogenesis, promotes lymphangiogenesis in CRC by mediating the VEGF-C expression.

VEGFC Predicts Poor Outcome in Pediatric as Well as Adult Acute Myeloid Leukemia: Insights in Associated Gene Expression Profiles.

Abstract 997Poster Board I-19A high vascular endothelial growth factor-C (VEGFC) mRNA level of primary blasts of pediatric Acute Myeloid Leukemia (AML) patients correlates with increased in vitro drug resistance and slow AML blast disappearance during induction therapy in vivo; measured by higher blast counts on day 15 in the bone marrow and a prolonged interval towards complete remission (HJM de Jonge et al. Clinical Cancer Research 2008).We set out to study both the effect of VEGFC on long term survival in AML and elucidate gene expression profiles associated with VEGFC expression by using Affymetrix HGU133Plus2.0 gene expression data of 100 pediatric and 525 adult AML patients.Pediatric AML patients with a high VEGFC mRNA expression level (i.e. above the median VEGFC expression level) showed a significantly shortened overall survival (P = .041, hazard ratio = 1,81). Among the adult cohort of 525 AML samples, patients with a high VEGFC mRNA level also had significantly reduced overall survival and event free survival (OS and EFS) compared to patients with a low VEGFC level (P < .001, hazard ratio = 1,41 for OS and P = .002, hazard ratio = 1,44 for EFS). The continuous variable VEGFC maintained its prognostic significance for OS and EFS in a multivariate cox-regression model after correction for possible confounding effects of age, white blood cell count, cytogenetic risk group (i.e. favorable, intermediate and unfavorable), NPM1 mutation and FLT3-ITD (P = .031, odds ratio 1.37 for OS and P = .008, odds ratio 1.42 for EFS).Differentially expressed probe sets were identified that distinguished patients with high from those with low VEGFC mRNA expression using a multivariate permutation test in Biometric Research Branch ArrayTools (BRB ArrayTools). Next, gene arrays of the pediatric AML cohort and a publicly available adult AML cohort of 180 gene arrays (Tomasson et al. Blood 2008) were used for validation of these probe sets. Finally, after two validation steps 569 probe sets were found to show differential high expression and 51 probe sets revealed differential low expression in AML with high mRNA levels of VEGFC, representative of distinctive biological processes (represented by GO-categories). Cell proliferation, VEGF receptor activity, signal transduction (e.g. PAK3 and SOS2 which are upstream activators of MEK1) and angiogenesis were upregulated, whereas genes associated with apoptosis (e.g. TIA1 and ANP32A) were found to be down regulated in AML with elevated VEGFC. In conclusion, high VEGFC mRNA expression levels are associated with reduced survival in adult and pediatric AML. Following the analysis of gene expression data in three independent AML cohorts the data suggest the involvement of specific biological processes in AML with (high) VEGFC expression with potential therapeutic impact. Disclosures:No relevant conflicts of interest to declare.

Expression of vascular endothelial growth factor C in pancreatic cancer and its effect upon lymph node metastasis

To investigate the expression of vascular endothelial growth factor C (VEGF-C) and the effect of antisense oligonucleotide (ASODN) upon lymph node metastasis of pancreatic cancer cell. We selected 15 cases of human pancreatic cancer and detected the expression of VEGF-C in primary tumor and lymph node metastasis tissues with immunohistochemistry. Meanwhile, the spontaneous lymphatic metastasis model in nude mice was established with orthotopic implantation for the human pancreatic cancer cell line PANC-1, isolation and culture of primary tumor and spontaneous lymphatic metastasis. The effect of VEGF-C special ASODN upon the apoptosis of pancreatic cancer cell derived from primary tumor and spontaneous lymphatic metastasis were detected by reverse transcription polymerase chain reaction (RT-PCR), enzyme linked immunosorbent assay (ELISA), flow cytometer and terminal deoxynucleotidyl transferase mediated-dUTP nick end labeling (TUNEL). In tissues of human pancreatic cancer, the values of VEGF-C on lymph nodes metastasis were more higher than primary tumor (P < 0.05). About the expression of VEGF-C on pancreatic cancer cell derived from spontaneous lymphatic metastasis and primary tumor in nude mice model, the mRNA levels of VEGF-C were 0.87 +/- 0.11 and 0.61 +/- 0.15 respectively, the VEGF-C levels in culture supernatants were (1682 +/- 157) pg/ml and (1404 +/- 128) pg/ml. The expression of VEGF-C on pancreatic cancer cells derived from lymphatic metastasis were also more higher than primary tumor (P < 0.05). In vitro and vivo, transfection of VEGF-C ASODN decreased the expression of VEGF-C in pancreatic cancer cell. In control group, scramble-sense oligonucleotide (SODN) group and ASODN group, the apoptosis rates of pancreatic cancer cells derived from lymph node metastasis were (2.8 +/- 1.0)%, (5.0 +/- 2.1)%, (13.2 +/- 2.2)% respectively in vitro, and were (1.8 +/- 0.5)%, (2.0 +/- 0.7)%, (4.4 +/- 1.0)% respectively in vivo, the apoptosis was increased significantly after transfection of VEGF-C ASODN (all P < 0.01). But pancreatic cancer cells derived from primary tumor were not effected (all P > 0.05). In human pancreatic cancer and nude mice model, the expression of VEGF-C on lymphatic metastasis was higher than primary tumor. The apoptosis of pancreatic cancer cells derived from spontaneous lymphatic metastasis were promoted by transfection of VEGF-C ASODN specially.

Inhibition of Cyclooxygenase-2 Suppresses Lymph Node Metastasis via Reduction of Lymphangiogenesis

Cyclooxygenase-2 (COX-2) inhibitor has been reported to suppress tumor progression. However, it is unclear whether this inhibitor can also prevent lymphatic metastasis. To determine the effects of COX-2 inhibitor on lymphatic metastasis, etodolac, a COX-2 inhibitor, was given p.o. to mice bearing orthotopic xenografts or with carcinomatous peritonitis induced with a highly metastatic human diffuse-type gastric carcinoma cell line, OCUM-2MLN. Tumor lymphangiogenesis was significantly decreased in etodolac-treated mice compared with control mice. Consistent with this decrease in lymphangiogenesis, the total weight of metastatic lymph nodes was less in etodolac-treated mice than in control mice. Immunohistochemical analysis revealed that the major source of vascular endothelial growth factor-C (VEGF-C) and VEGF-D was F4/80-positive macrophages in our models. The mRNA levels of VEGF-C in mouse macrophage-like RAW264.7 cells, as well as those in tumor tissues, were suppressed by etodolac. The growth of human dermal lymphatic microvascular endothelial cells was also suppressed by etodolac. Supporting these findings, etodolac also inhibited lymphangiogenesis in a model of chronic aseptic peritonitis, suggesting that COX-2 can enhance lymphangiogenesis in the absence of cancer cells. Our findings suggest that COX-2 inhibitor may be useful for prophylaxis of lymph node metastasis by reducing macrophage-mediated tumor lymphangiogenesis.

The balance of VEGF-C and VEGFR-3 mRNA is a predictor of lymph node metastasis in non-small cell lung cancer

A positive association between vascular endothelial growth factor-C (VEGF-C) expression and lymph node metastasis has been reported in several cancers. However, the relationship of VEGF-C and lymph node metastasis in some cancers, including non-small cell lung cancer (NSCLC), is controversial. We evaluated the VEGF-C and vascular endothelial growth factor receptor-3 (VEGFR-3) expression in NSCLC samples from patients who had undergone surgery between 1998 and 2002 using real-time quantitative RT–PCR and immunohistochemical staining. We failed to find a positive association between VEGF-C and VEGFR-3 mRNA expression and lymph node metastasis in NSCLC. An immunohistological study demonstrated that VEGF-C was expressed not only in cancer cells, but also in macrophages in NSCLC, and that VEGFR-3 was expressed in cancer cells, macrophages, type II pneumocytes and lymph vessels. The VEGF-C/VEGFR-3 ratio of the node-positive group was significantly higher than that of the node-negative group. Immunohistochemical staining showed that VEGFR-3 was mainly expressed in cancer cells. The immunoreactivity of VEGF-C and VEGFR-3 was roughly correlated to the mRNA levels of VEGF-C and VEGFR-3 in real-time PCR. VEGF-C mRNA alone has no positive association with lymph node metastasis in NSCLC. The VEGF-C/VEGFR-3 ratio was positively associated with lymph node metastasis in NSCLC. This suggests that VEGF-C promotes lymph node metastasis while being influenced by the strength of the VEGF-C autocrine loop, and the VEGF-C/VEGFR-3 ratio can be a useful predictor of lymph node metastasis in NSCLC.

Expression of Vascular Endothelial Growth Factor C and Vascular Endothelial Growth Factor Receptor 3 in Corneal Lymphangiogenesis

Lymphangiogenesis has been reported in vascularized corneas. However, the molecular mechanisms of lymphangiogenesis in the cornea are still unclear. Since lymphatic vessels may contribute to a decreased success rate of keratoplasty in vascularized cornea by accelerating antigen recognition and graft rejection, elucidation of the mechanisms of corneal lymphangiogenesis will facilitate the inhibition of lymphatic vessels and may improve the outcome of keratoplasty. This study aimed to examine the expression of vascular endothelial growth factor-C (VEGF-C), which is the only endogenous lymphangiogenic factor reported so far, and one of its receptors, vascular endothelial growth factor receptor-3 (VEGFR-3), in corneal lymphangiogenesis. A rat model was used in which silver nitrate application resulted in corneal circumferential neovascularization. The presence of lymphatic vessels in the rat injured cornea was examined with electron microscope. Corneal VEGF-C and VEGFR-3 mRNA levels were quantified with competitive reverse transcription polymerase chain reaction (RT-PCR), and VEGF-C and VEGFR-3 proteins were studied in situ using immunohistochemical analysis. Electron microscopy revealed lymphatic vessels in the vascularized rat corneas. Competitive RT-PCR demonstrated that the expression of VEGF-C mRNA in the rat cornea was normally absent, and was dramatically up-regulated 3 days after the injury, which gradually decreased. The VEGFR-3 expression in the rat cornea was minimally detected before the injury and was up-regulated 3 and 7 days after the injury. It was also minimally detected 2 and 4 weeks after the injury. In immunohistochemical analysis of the rat cornea 3 days after the injury, VEGF-C was mainly detected in inflammatory cells, and VEGFR-3 was demonstrated in several new vessels in the corneal stroma. These data suggest that VEGF-C and VEGFR-3 are pathophysiologically relevant endogenous factors in corneal lymphangiogenesis.