Expression Of VEGF-D Research Articles

Interleukin-8: A potent promoter of human lymphatic endothelial cell growth in gastric cancer.

Lymphatic metastasis is a major progression route of gastric cancer. Interleukin-8 (IL-8), as an inflammatory cytokine, is induced by Helicobacter pylori infection and is strongly associated with gastric cancer development and metastasis. The blood and lymphatic systems are similar in their function and gene expression profiles. It has been proposed that IL-8 activates angiogenesis. However, the direct role of IL-8 in lymphangiogenesis in gastric cancer remains unclear. We investigated the effect of IL-8 on the growth of human lymphatic endothelial cells (LECs). In addition, protein and mRNA expression of selected lymphangiogenesis markers was assessed in these cells. LECs were co-cultured with gastric cancer SGC7901 cells and exposed to various concentrations of IL-8 (0, 0.2, 0.5, 0.8 and 1.0 ng/ml). The Cell Counting Kit-8 was used to evaluate LEC proliferation (cultured for 1-6 days). Then, protein (immunofluorescence and western blotting) and mRNA [quantitative transcription-polymerase chain reaction (qPCR)] levels were measured in samples obtained from the 24-h cultured cells, for lymphatic vessel endothelial hyaluronic acid receptor-1 (LYVE-1), vascular endothelial growth factor (VEGF)-C, VEGF-D and vascular endothelial growth factor receptor-3 (VEGFR-3). The data presented herein demonstrated that IL-8 promotes the proliferation of LECs and enhances the protein and mRNA expression of LYVE-1. Notably, IL-8 inhibited VEGF-C, VEGF-D and VEGFR-3 protein expression as well as VEGF-D and VEGFR-3 mRNA expression. These findings suggest that IL-8 may be a potent inducer of LECs, although this effect does not appear to involve the VEGF-C/VEGF-D and VEGFR-3 signaling pathway.

0170 : VEGF-D translational regulations promotes tumor lymphatic vessels plasticity

The lymphatic vasculature has a major role in physiology. It drains the interstitial fluids and performs immune surveillance by transporting immunity cells. The lymphatic vessels are also involved in pathological conditions such as chronic inflammatory diseases, lymphedema and cancer. Lymphangiogenesis, the growth of new lymphatic vessels, is induced by the lymphangiogenic growth factors VEGF-C and VEGF-D. Recent studies suggest that VEGF-D is also involved in lymphatic dilatation through prostaglandin signaling pathways. In this study, we demonstrated the molecular mechanism involved in lymphatic dilatation. We identified a stress-induced translational regulation of VEGF-D expression through an IRES activation under heat shock conditions. We demonstrated that VEGF-D IRES activity was dependent of prostaglandin pathway as Cox-2 inhibitors are able to abolish the IRES-promoted VEGF-D protein synthesis, and lymphatic vessels dilatation. Using plasmon surface resonance on biotinylated VEGF-D mRNA coupled to mass spectrometry, we identified the IRES transacting factor (ITAF) that allows the recruitment of the ribosome to promote VEGF-D translational initiation: the Nucleolin. Our results bring new insights on the VEGF-D regulation and on the lymphatic vessels plasticity. As lymphatic vessels play a crucial role in inflammation by performing immune cells tissue trafficking, understanding the regulations of lymphatic vasodilatation is a crucial step toward an innovative inflammatory disease therapy.

Lymphangiogenesis in gastric cancer regulated through Akt/mTOR-VEGF-C/VEGF-D axis.

BackgroundLymphangiogenesis plays a significant role in metastasis and recurrence of gastric cancer. There is no report yet focusing on the modulation of VEGF pathway and lymphangiogenesis of gastric cancer by targeting Akt/mTOR pathway. This study aims to demonstrate the relationship between Akt/mTOR pathway and VEGF-C/-D in gastric cancer.MethodsWe collected surgically resected gastric adenocarcinoma specimens from 55 consented patients. Immunohistochemistry staining of p-Akt, p-mTOR, VEGF-C, VEGF-D were performed and scored by two independent pathologists. The results were presented as staining intensity and positive staining cell rate. We also measured lymphatic vessel density (LVD) by D2-40 staining. Different dosages of p-Akt inhibitor LY294002 (12.5 μM, 25 μM, 50 μM) and p-mTOR inhibitor Rapamycin (25 nM, 50 nM, 100 nM) were given to gastric cancer cell line SGC-7901 in vitro. The inhibition rate of cell growth was tested by MTT at 24 h, 48 h and 72 h, respectively and protein expressions of Akt, p-Akt, mTOR, p-mTOR, VEGF-C and VEGF-D were examined by Western blot.ResultsThe positive staining rates of p-Akt, p-mTOR, VEGF-C and VEGF-D in 55 gastric cancer clinical specimens were 74.54%, 85.45%, 72.73% and 58.18%. p-Akt and p-mTOR were positively correlated with VEGF-C and VEGF-D (p < 0.01). The LVD increased with incremental tendency of staining intensity of p-Akt, p-mTOR, VEGF-C and VEGF-D. LY294002 or Rapamycin significantly suppressed SGC-7901 cell growth and the inhibition rate was dose and time dependent (p < 0.001). In addition, the protein expression of p-Akt and p-mTOR were positively correlated with that of VEGF-C and VEGF-D (p < 0.05).ConclusionsThe level of LVD in gastric cancer specimens was significant higher than that of normal gastric tissue and was positively correlated with p-Akt, p-mTOR, VEGF-C and VEGF-D. Inhibition of p-Akt and p-mTOR, in vitro, decreased tumor cell VEGF-C and VEGF-D significantly. Therefore, we concluded that lymphangiogenesis of gastric cancer might be related to Akt/mTOR-VEGF-C/VEGF-D axis.

Angiotensin 1-7 Promotes Cardiac Angiogenesis Following Infarction

Angiogenesis is central to cardiac repair following myocardial infarction (MI). Cardiac angiotensin converting enzyme (ACE)2 significantly increased postMI, which is coincident with activated angiogenesis. The function of ACE2 is to generate angiotensin (Ang)1-7, an active peptide with cellular actions mediated by Mas receptors. The current study is to determine whether Ang(1-7) is involved in cardiac angiogenesis and facilitates cardiac repair. In the first portion of the study, the temporal expressions of cardiac ACE2 and Mas receptors were detected in rats with MI. In the second portion, MI rats were treated with or without a Mas receptor antagonist, A779 (1mg/kg/day given by minipump) for 7 days. Vascular density and expression of angiogenic mediators in the infarcted myocardium and cardiac function were examined. Compared to controls, ACE2 and Mas receptor levels were significantly increased in the infarcted myocardium for 4 weeks of the observation period. Newly formed vessels were evident in the infarcted myocardium at day 7. Mas receptor blockade significantly reduced vascular density in the infarcted myocardium and impaired ventricular function. In addition, A779 treatment significantly suppressed the cardiac expressions of vascular endothelial growth factor (VEGF)-D and matrix metalloproteinase (MMP)-9 but not expression of other angiogenic mediators, including monocyte Chemoattractant protein (MCP-1), VEGF-C, transforming growth factor (TGF)-β1 and integrin β3. These observations indicate that Ang(1-7) promotes angiogenesis via stimulating the expression of cardiac VEGF-D and MMP-9, thus facilitating cardiac repair and ventricular function.

VEGF-D as a marker in the aid of malignant metastatic pleural effusion diagnosis.

The sensitivity in cytology diagnosis of malignant metastatic pleural effusion (MMPE) is insufficient nowadays due to the similarity of the reactive mesothelial cells and malignant cells. Vascular endothelial growth factor (VEGF) is one of the key factors in tumor lymphangiogenesis and metastasis. Therefore, the aim of this study was to evaluate the value of VEGF and its homologs in the aid of MMPE diagnosis. A total of 217 pleural effusions samples were eligible for analysis. Among them, 81 malignant and 22 benign cases were made into the cell blocks for the immunocytochemical (ICC) staining of VEGF-A, VEGF-C, VEGF-D, VEGFR-2, and VEGFR-3 expression. Another 114 samples (41 malignant and 73 benign cases) were subjected to the ELISA test for the protein level of VEGF-D. In a total of 156 MMPE, only VEGF-D expression by ICC stain was significantly different between malignant (92.6%) and benign cases (9.1%) with P<0.001 in either nuclear or cytoplasmic staining. Only 6 malignant cases showed negative stain results. In addition, 3 of the 4 lung small cell carcinoma were immunoreactive for VEGF-D. However, some lymphocytes also showed nuclear staining pattern of VEGF-D. In contrast, the ELISA test for the VEGF-D protein levels failed to demonstrate the difference between malignant and benign pleural effusions. Among VEGF homologs, MMPE from various kinds of tumor origin, VEGF-D showed 92.6% rate of positive expression. ICC stain of VEGF-D is a useful marker in the aid of MMPE diagnosis.

Vascular endothelial growth factor receptor 3 signaling contributes to angioobliterative pulmonary hypertension.

The mechanisms involved in the development of severe angioobliterative pulmonary arterial hypertension (PAH) are multicellular and complex. Many of the features of human severe PAH, including angioobliteration, lung perivascular inflammation, and right heart failure, are reproduced in the Sugen 5416/chronic hypoxia (SuHx) rat model. Here we address, at first glance, the confusing and paradoxical aspect of the model, namely, that treatment of rats with the antiangiogenic vascular endothelial growth factor (VEGF) receptor 1 and 2 kinase inhibitor, Sugen 5416, when combined with chronic hypoxia, causes angioproliferative pulmonary vascular disease. We postulated that signaling through the unblocked VEGF receptor VEGFR3 (or flt4) could account for some of the pulmonary arteriolar lumen-occluding cell growth. We also considered that Sugen 5416-induced VEGFR1 and VEGFR2 blockade could alter the expression pattern of VEGF isoform proteins. Indeed, in the lungs of SuHx rats we found increased expression of the ligand proteins VEGF-C and VEGF-D as well as enhanced expression of the VEGFR3 protein. In contrast, in the failing right ventricle of SuHx rats there was a profound decrease in the expression of VEGF-B and VEGF-D in addition to the previously described reduction in VEGF-A expression. MAZ51, an inhibitor of VEGFR3 phosphorylation and VEGFR3 signaling, largely prevented the development of angioobliteration in the SuHx model; however, obliterated vessels did not reopen when animals with established PAH were treated with the VEGFR3 inhibitor. Part of the mechanism of vasoobliteration in the SuHx model occurs via VEGFR3. VEGFR1/VEGFR2 inhibition can be initially antiangiogenic by inducing lung vessel endothelial cell apoptosis; however, it can be subsequently angiogenic via VEGF-C and VEGF-D signaling through VEGFR3.

VEGF-C and VEGF-D expression and its correlation with lymph node metastasis in esophageal squamous cell cancer tissue.

To explore vascular endothelial growth factor C (VEGF-C) and VEGF-D expression and its correlation with lymph node metastasis in esophageal squamous cell cancer (ESCC) tissue. Immunohistochemical methods were applied to detect the levels of VEGF-C and VEGF-D expression in 64 surgicall removal ESCC tissues, tissues adjacent to cancer and normal tissues, and the relationship between VEGF-C and VEGF-D expression and lymph node metastasis was analyzed. Both VEGF-C and VEGF-D were expressed by varying degrees in esophageal cancer tissue, the tissue adjacent to cancer and normal tissue, and the positive expression rate went down successively. The positive expression rates of VEGF-C (59.4%) and VEGF-D (43.8%) in esophageal cancer tissue were significantly higher than in the tissue adjacent to cancer (34.4%, 15.6%) and normal tissue (20.3%, 12.5%), respectively, in which significant differences were manifested (p<0.01). Positive expression rates of VEGF-C and VEGF-D in esophageal cancers with lymph node metastasis were markedly higher than without such metastasis (p<0.01), while those in the tissue with TNM staging I~II were markedly lower than that with TNM staging III~IV (p<0.01). Both VEGF-C and VEGF-D are highly expressed in ESCC tissue, which may be related to the lymph node metastasis of cancer cells. Hence, VEGF-C and VEGF-D can be clinically considered as important reference indexes of lymph node metastasis in esophageal cancer.

Andrographolide decreased VEGFD expression in hepatoma cancer cells by inducing ubiquitin/proteasome-mediated cFos protein degradation

BackgroundAndrographolide (Andro) is reported to inhibit hepatoma tumor growth in our previous studies. This study aims to further search the critical signals involved in such Andro-induced inhibition. MethodsThe anti-tumor effect of Andro was evaluated in vivo. Cancer PathwayFinder RT2 Profiler™ PCR array was used to find the altered genes. Real-time PCR was used to detect the mRNA expression. Protein expression was detected by Western-blot analysis, enzyme-linked immunosorbent assay (ELISA) and immunohistochemical staining. Activator protein-1 (AP-1) transcriptional activity was detected by luciferase reporter assay. ResultsAndro (10mg/kg) inhibited hepatoma tumor growth in vivo. The expression of four genes decreased in Andro-treated tumor tissues. Among which, vascular endothelial growth factor (VEGFD) was the highest decreased gene. The decreased VEGFD expression was further confirmed by real-time PCR and immunohistochemical staining assay. Andro decreased VEGFD mRNA and protein expression in hepatoma Hep3B and HepG2 cells. Andro also decreased VEGFD amount in Hep3B cell supernatant. Andro decreased cFos protein expression and its translocation into nucleus, and also reduced AP-1 luciferase activity. Further results showed that Andro induced polyubiquitination of cFos. Proteasome inhibitor MG132 reversed the decreased expression of cFos protein, and the decreased mRNA and protein expression of VEGFD. SP600125, an inhibitor of c-Jun N-terminal kinase (JNK), reversed the decreased expression of cFos and VEGFD induced by Andro. ConclusionsAndro decreased VEGFD expression in hepatoma cancer cells via inducing c-fos protein degradation, which will contribute to its anti-cancer activity, and JNK plays some roles in regulating this process.

Norcantharidin Inhibits Lymphangiogenesis by Downregulating the Expression of VEGF-C and VEGF-D in Human Dermal Lymphatic Endothelial Cells in vitro

Aims: To investigate the effects of norcantharidin on the growth and migration of human dermal lymphatic endothelial cells (HDLECs) and further characterize its effect on lymphangiogenesis. Methods: A 3-dimensional fibrin gel lymphangiogenesis model was built. Flow cytometry was used to analyze the rate of apoptosis and necrosis. RT-PCR, immunohistochemistry and immunoblotting assays were used to examine the effect of norcantharidin on vascular endothelial growth factor C (VEGF-C), VEGF-D and VEGF receptor 3 during in vitro lymphangiogenesis. Results: Norcantharidin caused a marked dose and time-dependent inhibition of the growth of HDLECs with an IC₅₀ of 40 nmol/l. The apoptotic rate of HDLECs was 13.21 ± 1.60% 24 h after treatment with 7.5 nmol/l norcantharidin and 42.34 ± 3.80% with 90 nmol/l norcantharidin (p < 0.01 vs. controls in both). Fibrin gel assays showed that norcantharidin (15 nmol/l) reduced the number of tubular structures from 68.4 ± 5.2 in untreated controls to 10.9 ± 2.3 (p = 0.000). RT-PCR, immunohistochemistry and immunoblotting assays showed norcantharidin markedly reduced the expression of VEGF-C and VEGF-D. Conclusion: Norcantharidin inhibits lymphangiogenesis by downregulating the expression of VEGF-C and VEGF-D, suggesting that norcantharidin could be an effective agent for targeting neolymphangiogenesis.

Correlation Analysis of Nasopharyngeal Carcinoma TNM Staging with Serum EA IgA and VCA IgA in EBV and VEGF-C and -D.

BackgroundNasopharyngeal carcinoma often occurs in humans in the nasopharyngeal epithelium area. Ebstein-Barr (EB) virus plays a key role in the process of nasopharyngeal carcinoma lesions. Early antigen antibody (EA-IgA) and viral capsid antigen IgA (VCA-IgA) of EB virus detection in serum can effectively monitor the process of nasopharyngeal carcinoma lesions. Serum vascular endothelial growth factor (VEGF) -C and VEGF-D expression detection can reflect the distant metastases ability of human tumor cells.Material/Methods153 cases of nasopharyngeal carcinoma patients in our hospital were enrolled, while 148 cases of healthy adults were selected as control. ELISA was used to detect serum EA-IgA, VCA-IgA, VEGF-C and -D expression levels. Spearman rank correlation analysis was applied to test the correlation of nasopharyngeal carcinoma TNM clinical stage and different indexes.ResultsSerum EA-IgA, VCA-IgA, VEGF-C and -D expression in nasopharyngeal carcinoma patients was 43.74±2.6 U·mL−1, 62.5±2.7 U·mL−1, 473.25±3.4 pg·mL−1, and 498.36±2.3 pg·mL−1, respectively, which was significantly higher than in the control group as 18.65±3.7 U·mL−1, 23.74±1.5 U·mL−1, 225.42±2.3 pg·mL−1, and 257.24±3.5 pg·mL−1 (P<0.05). Nasopharyngeal carcinoma TNM clinical staging was obviously correlated with serum EA-IgA, VCA-IgA, and VEGF-C (P<0.05), but not VEGF-D (P>0.05).ConclusionsNasopharyngeal carcinoma patient serum EA-IgA and VCA-IgA expression levels were significantly correlated with TNM staging. The high levels of these 3 indicators suggest advanced nasopharyngeal carcinoma TNM staging and serious lesions.

Kaposi's Sarcoma-Associated Herpesvirus Induces Nrf2 during De Novo Infection of Endothelial Cells to Create a Microenvironment Conducive to Infection

Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiological agent of Kaposi's sarcoma (KS) and primary effusion B-cell lymphoma. KSHV induces reactive oxygen species (ROS) early during infection of human dermal microvascular endothelial (HMVEC-d) cells that are critical for virus entry. One of the downstream targets of ROS is nuclear factor E2-related factor 2 (Nrf2), a transcription factor with important anti-oxidative functions. Here, we show that KS skin lesions have high Nrf2 activity compared to healthy skin tissue. Within 30 minutes of de novo KSHV infection of HMVEC-d cells, we observed Nrf2 activation through ROS-mediated dissociation from its inhibitor Keap1, Ser-40 phosphorylation, and subsequent nuclear translocation. KSHV binding and consequent signaling through Src, PI3-K and PKC-ζ were also important for Nrf2 stability, phosphorylation and transcriptional activity. Although Nrf2 was dispensable for ROS homeostasis, it was essential for the induction of COX-2, VEGF-A, VEGF-D, Bcl-2, NQO1, GCS, HO1, TKT, TALDO and G6PD gene expression in KSHV-infected HMVEC-d cells. The COX-2 product PGE2 induced Nrf2 activity through paracrine and autocrine signaling, creating a feed-forward loop between COX-2 and Nrf2. vFLIP, a product of KSHV latent gene ORF71, induced Nrf2 and its target genes NQO1 and HO1. Activated Nrf2 colocalized with the KSHV genome as well as with the latency protein LANA-1. Nrf2 knockdown enhanced ORF73 expression while reducing ORF50 and other lytic gene expression without affecting KSHV entry or genome nuclear delivery. Collectively, these studies for the first time demonstrate that during de novo infection, KSHV induces Nrf2 through intricate mechanisms involving multiple signal molecules, which is important for its ability to manipulate host and viral genes, creating a microenvironment conducive to KSHV infection. Thus, Nrf2 is a potential attractive target to intervene in KSHV infection and the associated maladies.

Proteins of the VEGFR and EGFR pathway as predictive markers for adjuvant treatment in patients with stage II/III colorectal cancer: results of the FOGT-4 trial.

BackgroundUnlike metastatic colorectal cancer (CRC) there are to date few reports concerning the predictive value of molecular biomarkers on the clinical outcome in stage II/III CRC patients receiving adjuvant chemotherapy. Aim of this study was to assess the predictive value of proteins related with the EGFR- and VEGFR- signalling cascades in these patients.MethodsThe patients' data examined in this study were from the collective of the 5-FU/FA versus 5-FU/FA/irinotecan phase III FOGT-4 trial. Tumor tissues were stained by immunohistochemistry for VEGF-C, VEGF-D, VEGFR-3, Hif-1 α, PTEN, AREG and EREG expression and evaluated by two independent, blinded investigators.Survival analyses were calculated for all patients receiving adjuvant chemotherapy in relation to expression of all makers above.ResultsPatients with negative AREG and EREG expression on their tumor had a significant longer DFS in comparison to AREG/EREG positive ones (p< 0.05). The benefit on DFS in AREG-/EREG- patients was even stronger in the group that received 5-FU/FA/irinotecan as adjuvant treatment (p=0.002). Patients with strong expression of PTEN profited more in terms of OS under adjuvant treatment containing irinotecan (p< 0.05). Regarding markers of the VEGFR- pathway we found no correlation of VEGF-C- and VEGFR-3 expression with clinical outcome. Patients with negative VEGF-D expression had a trend to live longer when treated with 5-FU/FA (p=0.106). Patients who were negative for Hif-1 α, were disease-free in more than 50% at the end of the study and showed significant longer DFS-rates than those positive for Hif-1 α (p=0.007). This benefit was even stronger at the group treated with 5-FU/FA/irinotecan (p=0.026). Finally, AREG-/EREG-/PTEN+ patients showed a trend to live longer under combined treatment combination.ConclusionsThe addition of irinotecan to adjuvant treatment with 5-FU/FA does not provide OS or DFS benefit in patients with stage II/III CRC. Nevertheless, AREG/EREG negative, PTEN positive and Hif-1 α negative patients might profit significantly in terms of DFS from a treatment containing fluoropyrimidines and irinotecan. Our results suggest a predictive value of these biomarkers concerning adjuvant chemotherapy with 5-FU/FA +/− irinotecan in stage II/III colorectal cancer.Electronic supplementary materialThe online version of this article (doi:10.1186/s13046-014-0083-8) contains supplementary material, which is available to authorized users.

Head and neck squamous cell carcinoma lymphatic spread and survival: Relevance of vascular endothelial growth factor family for tumor evaluation.

Head and neck squamous cell carcinoma (HNSCC) is primarily a locoregional disease in which the cervical lymph nodes are the chief site of metastasis. The purpose of this study was to examine the relationship between lymphangiogenesis and clinicopathological aspects of HNSCC and its metastasis. Fifty-two patients with HNSCC and metastatic lymph nodes from 21 of these subjects were analyzed by immunohistochemistry. The HNSCC samples were predominantly negative for vascular endothelial growth factor (VEGF)-C, VEGF-D, and vascular endothelial growth factor receptor (VEGFR)3. There was an association between the density of lymph vessels (measured by D2-40 staining) in the lymph nodes and advanced-stage tumors. There was no link between the expression of these proteins and survival rates. Although lymphatic spread is a significant event in the progression of HNSCC, the expression of VEGF-C, VEGF-D, and VEGFR3 does not correlate with clinicopathological characteristics, suggesting that other signaling pathways mediate lymphangiogenesis in HNSCC.

MiR-886-5p Inhibition Inhibits Growth and Induces Apoptosis of MCF7 Cells

To explore the molecular mechanisms of miR-886-5p in breast cancer., we examined roles in inhibiting growth and migration of MCF-7 cells. MiR-886-5p mimics and inhibitors were used to express or inhibit MiR-886-5p, respectively, and MTT and clone formation assays were used to determine the survival and proliferation. Hoechst 33342/ PI double staining was applied to detect apoptosis. The expression of caspase-3, caspase-8, caspase-9, MT1-MMP, VEGF-C and VEGF-D was detected by Western blotting, and the levels of MMP2 and MMP9 secreted from MCF-7 cells were assessed by ELISA. MCF-7 cell migration was determined by wound healing and Transwell assays. We found that the growth of MCF-7 cells was inhibited upon decreasing miR-886-5p levels. Inhibiting miR-866-5p also significantly induced apoptosis and decreased the migratory capacity of these cells. The expression of VEGF-C, VEGF-D, MT1-MMP, MMP2, and MMP9 was also found to be decreased as compared to controls. Our data show that downregulation of miR-886-5p expression in MCF-7 cells could significantly inhibit cell growth and migration. This might imply that inhibiting miR-886-5p could be a therapeutic strategy in breast cancer.

VEGF-D-induced draining lymphatic enlargement and tumor lymphangiogenesis promote lymph node metastasis in a xenograft model of ovarian carcinoma

BackgroundVascular 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.MethodsHuman 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.ResultsMice 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.ConclusionsOur 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.

Expression of Tiam1, C-met and VEGF-D in colorectal carcinoma

Expression of Tiam1, C-met and VEGF-D in colorectal carcinoma

Transforming Growth Factor-β1 Downregulates Vascular Endothelial Growth Factor-D Expression in Human Lung Fibroblasts via the Jun NH2-Terminal Kinase Signaling Pathway

Vascular endothelial growth factor (VEGF)-D, a member of the VEGF family, induces both angiogenesis and lymphangiogenesis by activating VEGF receptor-2 (VEGFR-2) and VEGFR-3 on the surface of endothelial cells. Transforming growth factor (TGF)-β1 has been shown to stimulate VEGF-A expression in human lung fibroblast via the Smad3 signaling pathway and to induce VEGF-C in human proximal tubular epithelial cells. However, the effects of TGF-β1 on VEGF-D regulation are unknown. To investigate the regulation of VEGF-D, human lung fibroblasts were studied under pro-fibrotic conditions in vitro and in idiopathic pulmonary fibrosis (IPF) lung tissue. We demonstrate that TGF-β1 downregulates VEGF-D expression in a dose- and time-dependent manner in human lung fibroblasts. This TGF-β1 effect can be abolished by inhibitors of TGF-β type I receptor kinase and Jun NH2-terminal kinase (JNK), but not by Smad3 knockdown. In addition, VEGF-D knockdown in human lung fibroblasts induces G1/S transition and promotes cell proliferation. Importantly, VEGF-D protein expression is decreased in lung homogenates from IPF patients compared with control lung. In IPF lung sections, fibroblastic foci show very weak VEGF-D immunoreactivity, whereas VEGF-D is abundantly expressed within alveolar interstitial cells in control lung. Taken together, our data identify a novel mechanism for downstream signal transduction induced by TGF-β1 in lung fibroblasts, through which they may mediate tissue remodeling in IPF.

Expression and clinical significance of neuropilin-2 in colorectal cancer: correlation with lymphangiogenic factors

Purpose: Neuropilin-2 is expressed by venous and lymphatic endothelial cells and can bind the lymphangiogenesis-associated ligand vascular endothelial growth factor (VEGF)-C and D. Recently, neuropilin-2 was shown to play a role in lymph node metastasis and cancer promotion. The aim of this study was to determine the clinical relevance of neuropilin-2 expression in colorectal carcinomas. Methods: Immunohistochemical analysis of neuropilin-2 expression of 350 colorectal cancer samples was evaluated using tissue microarray. The relationship between neuropilin-2 expression and other clinicopathological variables were analyzed in colorectal carcinomas. To clarify the role of neuropilin-2 in colorectal cancer lymphangiogenesis, its expression was examined with VEGF-C, VEGF-D, and VEGF receptor 3 expression of the colorectal carcinomas. Results: Neuropilin-2 was detected in 176 of 350 colorectal carcinomas (50.3%). Overexpression of neuropilin-2 was correlated with lymph node metastasis (P=0.041) and VEGF-D expression (P=0.020), but was not correlated with TNM (tumor, node, metastasis) stage, other lymphangiogenic factor expression and survival colorectal cancer patients. Conclusion: Neuropilin-2 expression was correlated with lymph node metastasis and VEGF-D expression in colorectal carcinomas. Our results suggest a possible role of neuropilin-2 in lymphangiogenesis and cancer progression in colorectal carcinomas.

Correlation between vascular endothelial growth factors and their receptor with lymph node metastasis of lung cancer

Objective To investigate the expression of vascular endothelial growth factor-C (VEGF-C),VEGF-D and VEGF receptor-3 (VEGFR-3) in non-small cell lung cancer (NSCLC) and their correlation with lymph node metastasis.Methods By using fluorescence quantitative RT-PCR,the expression of VEGF-C,VEGF-D and VEGFR-3 was detected in 96 patients with NSCLC.Results There was a significant correlation betwwen the positive expression of VEGF-C,VEGF-D and VEGFR-3 and lymph node metastasis and TNM stage (P ＜ 0.05).The expression levels of VEGF-C,VEGF-D and VEGFR-3 mRNA in lung cancer tissues were significantly higher than in normal tissues (P ＜0.01).in NSCLC,there was a positive correlation among VEGF-C,VEGF-D and VEGFR-3 mRNA expression.Conclusion VEGF-C,VEGF-D and VEGF-3 play an important role in the occurrence,development and lymph node metastasis of NSCLC. Key words: Lung cancer; Vascular endothelial growth factor; Lymph node; Metastasis

Neither expression of VEGF-C/D nor lymph vessel density supports lymphatic invasion as the mechanism responsible for local spread of recurrent salivary pleomorphic adenoma

Recent research suggests that multinodular recurrent pleomorphic adenoma (PA) might result from cell migration through lymphatics. Lymphangiogenesis in malignancies is mediated by vascular endothelial growth factors C and D (VEGF-C/D). We studied the expression of VEGF-C/D in PA by immunohistochemistry as well as lymphatic vessel density (LVD). In 6 non-recurrent, 4 primary-to-recur, and 10 recurrent PAs, VEGF-C/D expression was assessed by immunohistochemistry. Staining was scored in terms of staining intensity (0 = absent to 3 = strong), and the percentage of positive tumor cells (scored as 0 (0-19 %), 1 (20-39 %), 2 (40-50 %), and 3 (60-100 %)) and a sum score were calculated. Intra- and peritumoral LVD was assessed by counting of LV after immunostaining, using the D2-40 antibody. All but one sample were VEGF-C negative. The differences in VEGF-D expression between non-recurrent, primary-to-recur, and recurrent PAs were not significant (p>0.05). VEGF-D expression did not correlate with peritumoral LVD (p>0.05). Our study revealed a significant difference between intra- and peritumoral LVD values when comparing individual and all sample groups (p=0.01). The lack of VEGF-C expression and of significant differences in VEGF-D expression and peritumoral LVD between patients with non-recurrent, primary-to-recur, and recurrent PAs does not support the lymphangiogenic local spread hypothesis