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

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

Peritumoral lymphangiogenesis induced by vascular endothelial growth factor C and D promotes lymph node metastasis in breast cancer patients

ABSTRACT: 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.     

Periostin directly and indirectly promotes tumor lymphangiogenesis of head and neck cancer

Background

Metastasis to regional lymph nodes via lymphatic vessels plays a key role in cancer progression. Tumor lymphangiogenesis is known to promote lymphatic metastasis, and vascular endothelial growth factor C (VEGF-C) is a critical activator of tumor lymphangiogenesis during the process of metastasis. We previously identified periostin as an invasion- and angiogenesis-promoting factor in head and neck squamous cell carcinoma (HNSCC). In this study, we discovered a novel role for periostin in tumor lymphangiogenesis.

Methods and Findings

Periostin overexpression upregulated VEGF-C mRNA expression in HNSCC cells. By using conditioned media from periostin-overexpressing HNSCC cells, we examined tube formation of lymphatic endothelial cells. Conditioned media from periostin-overexpressing cells promoted tube formation. To know the correlation between periostin and VEGF-C, we compared Periostin expression with VEGF-C expression in 54 HNSCC cases by immunohistochemistry. Periostin expression was correlated well with VEGF-C expression in HNSCC cases. Moreover, correlation between periostin and VEGF-C secretion was observed in serum from HNSCC patients. Interestingly, periostin itself promoted tube formation of lymphatic endothelial cells independently of VEGF-C. Periostin-promoted lymphangiogenesis was mediated by Src and Akt activity. Indeed possible correlation between periostin and lymphatic status in periostin-overexpressing xenograft tumors and HNSCC cases was observed.

Conclusions

Our findings suggest that periostin itself as well as periostin-induced upregulation of VEGF-C may promote lymphangiogenesis. We suggest that periostin may be a marker for prediction of malignant behaviors in HNSCC and a potential target for future therapeutic intervention to obstruct tumoral lymphatic invasion and lymphangiogenesis in HNSCC patients.     

Effects of acute exercise, exercise training, and diabetes on the expression of lymphangiogenic growth factors and lymphatic vessels in skeletal muscle

Blood and lymphatic vessels together form the circulatory system, allowing the passage of fluids and molecules within the body. Recently we showed that lymphatic capillaries are also found in the capillary bed of skeletal muscle. Exercise is known to induce angiogenesis in skeletal muscle, but it is not known whether exercise has effects on lymphangiogenesis or lymphangiogenic growth factors. We studied lymphatic vessel density and expression of the main lymphangiogenic growth factors VEGF-C and VEGF-D and their receptor VEGFR-3 in response to acute running exercise and endurance exercise training in the skeletal muscle of healthy and diabetic mice. VEGF-C mRNA expression increased after the acute exercise bout (P < 0.05) in healthy muscles, but there was no change in diabetic muscles. VEGF-C levels were not changed either in healthy or in diabetic muscle after the exercise training. Neither acute exercise nor exercise training had an effect on the mRNA expression of VEGF-D or VEGFR-3 in healthy or diabetic muscles. Lymphatic vessel density was similar in sedentary and trained mice and was >10-fold smaller than blood capillary density. Diabetes increased the mRNA expression of VEGF-D (P < 0.01). Increased immunohistochemical staining of VEGF-D was found in degenerative muscle fibers in the diabetic mice. In conclusion, the results suggest that acute exercise or exercise training does not significantly affect lymphangiogenesis in skeletal muscle. Diabetes increased the expression of VEGF-D in skeletal muscle, and this increase may be related to muscle fiber damage.     

Tumor-induced lymphangiogenesis: a target for cancer therapy?

Recent advances in understanding the biology of lymphangiogenesis, the new growth of lymphatic vessels, have cast new light on the molecular basis of metastasis to regional lymph nodes. The receptor tyrosine kinase VEGFR-3 is virtually exclusively expressed on lymphatic but not blood endothelium in the adult, and activation of VEGFR-3 by its ligands VEGF-C and VEGF-D is sufficient to induce lymphangiogenesis. Correlative studies with human tumors and functional studies using animal tumor models show that increased levels of VEGF-C or VEGF-D in tumors lead to enhanced numbers of lymphatic vessels in the vicinity of tumors, which in turn promotes metastasis to regional lymph nodes by providing a greater number of entry sites into the lymphatic system for invading tumor cells. These findings have prompted studies to investigate whether inhibitors of VEGFR-3 activation might represent novel therapeutic agents for the suppression of metastasis. However, a number of points regarding the therapeutic potential of anti-lymphangiogenic treatments in the context of cancer remain to be addressed. The spectrum and relative importance of molecules that induce lymphangiogenesis and the regulation of their expression during tumor progression, the reversibility of tumor-induced lymphangiogenesis, and possible side-effects of anti-lymphangiogenesis-based therapies all need to be investigated. Most importantly, the extent to which lymph node metastases contribute to the formation of metastases in other organs remains to be elucidated. These aspects are the focus of this review, and their investigation should serve as a roadmap to possible translational application.     

Phage-Derived Fully Human Monoclonal Antibody Fragments to Human Vascular Endothelial Growth Factor-C Block Its Interaction with VEGF Receptor-2 and 3

Vascular endothelial growth factor C (VEGF-C) is a key mediator of lymphangiogenesis, acting via its receptors VEGF-R2 and VEGF-R3. High expression of VEGF-C in tumors correlates with increased lymphatic vessel density, lymphatic vessel invasion, sentinel lymph node metastasis and poor prognosis. Recently, we found that in a chemically induced skin carcinoma model, increased VEGF-C drainage from the tumor enhanced lymphangiogenesis in the sentinel lymph node and facilitated metastatic spread of cancer cells via the lymphatics. Hence, interference with the VEGF-C/VEGF-R3 axis holds promise to block metastatic spread, as recently shown by use of a neutralizing anti-VEGF-R3 antibody and a soluble VEGF-R3 (VEGF-C/D trap). By antibody phage-display, we have developed a human monoclonal antibody fragment (single-chain Fragment variable, scFv) that binds with high specificity and affinity to the fully processed mature form of human VEGF-C. The scFv binds to an epitope on VEGF-C that is important for receptor binding, since binding of the scFv to VEGF-C dose-dependently inhibits the binding of VEGF-C to VEGF-R2 and VEGF-R3 as shown by BIAcore and ELISA analyses. Interestingly, the variable heavy domain (V_H) of the anti-VEGF-C scFv, which contains a mutation typical for camelid heavy chain-only antibodies, is sufficient for binding VEGF-C. This reduced the size of the potentially VEGF-C-blocking antibody fragment to only 14.6 kDa. Anti-VEGF-C V_H-based immunoproteins hold promise to block the lymphangiogenic activity of VEGF-C, which would present a significant advance in inhibiting lymphatic-based metastatic spread of certain cancer types.     

Lymphangiogenesis and tumor metastasis

The lymphatic system transports interstitial fluid and macromolecules from tissues back to the blood circulation, and plays an important role in the immune response by directing the traffic of lymphocytes and antigen-presenting cells. The lymphatic system also constitutes one of the most important pathways of tumor dissemination. In many human cancers, increased expression of vascular endothelial growth factor-C (VEGF-C) is correlated with regional lymph node metastases. Experimental studies using transgenic mice overexpressing VEGF-C or xenotransplantation of VEGF-C-expressing tumor cells into immunodeficient mice have demonstrated a role for VEGF-C in tumor lymphangiogenesis and the subsequent formation of lymph node metastases. However, there is at present little evidence for lymphangiogenesis in human tumors and the relative importance of preexisting vs. newly formed lymphatics for metastasis in humans remains to be determined. Nonetheless, the striking correlation between the levels of VEGF-C in primary human tumors and lymph node metastases predicts its importance in cancer spread. Aside from promoting lymphangiogenesis, VEGF-C may also activate lymphatics to promote tumor cell chemotaxis, lymphatic intravasation and hence tumor cell dissemination.Work in the authors' laboratories was supported by grants from the Swiss National Science Foundation (no. 3100–064037.00) (to M.S.P), the Speaker's Fund for Biomedical Research (to M.S.) and the Peter Sharp Foundation (to M.S.). Parts of this review will be published in abbreviated form in Thrombosis and Haemostasis     

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

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

Tumor lymphangiogenesis and melanoma metastasis

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

Multiple Analyses of G-Protein Coupled Receptor (GPCR) Expression in the Development of Gefitinib-Resistance in Transforming Non-Small-Cell Lung Cancer

There is increasing evidence that functional crosstalk between GPCRs and EGFR contributes to the progression of colon, lung, breast, ovarian, prostate and head and neck tumors. In this study, we performed multiple analyses of GPCR expression in a gefitinib-resistant non-small cell lung cancer (NSCLC) cell line, H1975, which harbors an L858R/T790M mutation. To determine the expression profile of mRNAs encoding 384 GPCRs in normal human lung fibroblast (NHLF) and H1975 cells, a GPCR-specific microarray analysis was performed. A heat-map of the microarray revealed considerable differences in the expression of GPCRs between NHLF and H1975 cells. From the GPCR expression list, we selected some GPCR agonists/antagonist to investigate whether the respective ligands could affect the growth of H1975 cells. Among them, treatment with either a selective antagonist of adenosine A2a receptors, which were highly expressed in H1975 cell and another gefitinib-resistant NSCLC cells, HCC827GR cells or “small interfering RNA” (siRNA) targeting adenosine A2a receptors produced a significant decrease in cell viability of both H1975 and HCC827GR cells. Among up-regulated GPCRs in H1975 cells, Gs-, Gi- and Gq-coupled GPCRs were expressed almost equally. Among down-regulated GPCRs, Gi-coupled GPCRs were dominantly expressed in H1975 cells. The present results suggest that multilayered crosstalk between GPCRs and EGFR may play an important role in orchestrating downstream signaling molecules that are implicated in the development of gefitinib-resistant NSCLC.     

HIF-1α coordinates lymphangiogenesis during wound healing and in response to inflammation

This study aimed to investigate the mechanisms that coordinate lymphangiogenesis. Using mouse models of lymphatic regeneration and inflammatory lymphangiogenesis, we explored the hypothesis that hypoxia inducible factor-α (HIF-1α) is a central regulator of lymphangiogenesis. We show that HIF-1α inhibition by small molecule inhibitors (YC-1 and 2-methyoxyestradiol) results in delayed lymphatic repair, decreased local vascular endothelial growth factor-C (VEGF-C) expression, reduced numbers of VEGF-C(+) cells, and reductions in inflammatory lymphangiogenesis. Using transgenic HIF-1α/luciferase mice to image HIF-1α expression in real time in addition to Western blot analysis and pimonidazole staining for cellular hypoxia, we demonstrate that hypoxia stabilizes HIF-1α during initial stages of wound repair (1-2 wk); whereas inflammation secondary to gradients of lymphatic fluid stasis stabilizes HIF-1α thereafter (3-6 wk). In addition, we show that CD4(+) cell-mediated inflammation is necessary for this response and regulates HIF-1α expression by macrophages, as CD4-deficient or CD4-depleted mice demonstrate 2-fold reductions in HIF-1α expression as compared to wild-types. In summary, we show that HIF-1α is a critical coordinator of lymphangiogenesis by regulating the expression of lymphangiogenic cytokines as part of an early response mechanism to hypoxia, inflammation, and lymphatic fluid stasis.     

Differential mRNA and tissue expression of lymphangiogenic growth factors (VEGF-C and -D) and their receptor (VEGFR-3) during tail regeneration in a gecko

Lymphangiogenesis, the growth of new lymph vessels, has important roles in both normal and pathological lymphatic function. Despite recent advances, the precise molecular mechanisms behind the lymphangiogenic process remain unclear. The Australian marbled gecko, Christinus marmoratus, voluntarily drops its tail (autotomy) as a predator avoidance strategy. Following autotomy a new tail is regenerated including lymphatic drainage pathways. We examined the molecular control of lymphangiogenesis within the unique model of the regenerating gecko tail. Partial sequences were obtained of the gecko lymphangiogenic growth factors, vascular endothelial growth factor C (VEGF-C) and VEGF-D along with their receptor VEGFR-3. These were highly homologous to other vertebrates. Quantitative real-time polymerase chain reaction (PCR) demonstrated up-regulation of VEGF-C, VEGF-D and VEGFR-3 mRNA expression during the early and middle stages of tail regeneration (between 4 and 9 weeks following autotomy), in late regeneration (12 weeks) and during mid-regeneration (7 and 9 weeks), respectively. VEGF-C and VEGF-D immunostaining was observed lining some lymphatic-like and blood vessels in early–mid tail regeneration, indicating possible associations of the proteins with VEGFRs on endothelia. Keratinocytes and fibroblasts also showed positive staining of VEGF-C and VEGF-D in early–mid tail regeneration. Additionally, VEGF-C was localised in adipose tissue in all tail states examined. This work suggests that specific timings exist for the expression of the lymphangiogenic growth factors, VEGF-C and VEGF-D, and their receptor, VEGF-R3, throughout the regeneration of a functional lymphatic network. Along with localisation data, this suggests potential functions for the growth factors in lymphangiogenesis and angiogenesis throughout tail regeneration.     

miR-146a Inhibits Cell Growth,Cell Migration and Induces Apoptosis in Non-Small Cell Lung Cancer Cells

Aberrant expression of microRNA-146a (miR-146a) has been reported to be involved in the development and progression of various types of cancers. However, its role in non-small cell lung cancer (NSCLC) has not been elucidated. The aim of this study was to investigate the contribution of miR-146a to various aspects of the malignant phenotype of human NSCLCs. In functional experiments, miR-146a suppressed cell growth, induced cellular apoptosis and inhibited EGFR downstream signaling in five NSCLC cell lines (H358, H1650, H1975, HCC827 and H292). miR-146a also inhibited the migratory capacity of these NSCLC cells. On the other hand, miR-146a enhanced the inhibition of cell proliferation by drugs targeting EGFR, including both TKIs (gefitinib, erlotinib, and afatinib) and a monoclonal antibody (cetuximab). These effects were independent of the EGFR mutation status (wild type, sensitizing mutation or resistance mutation), but were less potent compared to the effects of siRNA targeting of EGFR. Our results suggest that these effects of miR-146a are due to its targeting of EGFR and NF-κB signaling. We also found, in clinical formalin fixed paraffin embedded (FFPE) lung cancer samples, that low expression of miR-146a was correlated with advanced clinical TNM stages and distant metastasis in NSCLC (P<0.05). The patients with high miR-146a expression in their tumors showed longer progression-free survival (25.6 weeks in miR-146a high patients vs. 4.8 weeks in miR-146a low patients, P<0.05). miR-146a is therefore a strong candidate prognostic biomarker in NSCLC. Thus inducing miR-146a might be a therapeutic strategy for NSCLC.     

LPA1/3 signaling mediates tumor lymphangiogenesis through promoting CRT expression in prostate cancer

Lysophosphatidic acid (LPA) is a bioactive lipid growth factor which is present in high levels in serum and platelets. LPA binds to its specific G-protein-coupled receptors, including LPA₁ to LPA₆, thereby regulating various physiological functions, including cancer growth, angiogenesis, and lymphangiogenesis. Our previous study showed that LPA promotes the expression of the lymphangiogenic factor vascular endothelial growth factor (VEGF)-C in prostate cancer (PCa) cells. Interestingly, LPA has been shown to regulate the expression of calreticulin (CRT), a multifunctional chaperone protein, but the roles of CRT in PCa progression remain unclear. Here we investigated the involvement of CRT in LPA-mediated VEGF-C expression and lymphangiogenesis in PCa. Knockdown of CRT significantly reduced LPA-induced VEGF-C expression in PC-3 cells. Moreover, LPA promoted CRT expression through LPA receptors LPA₁ and LPA₃, reactive oxygen species (ROS) production, and phosphorylation of eukaryotic translation initiation factor 2α (eIF2α). Tumor-xenografted mouse experiments further showed that CRT knockdown suppressed tumor growth and lymphangiogenesis. Notably, clinical evidence indicated that the LPA-producing enzyme autotaxin (ATX) is related to CRT and that CRT level is highly associated with lymphatic vessel density and VEGF-C expression. Interestingly, the pharmacological antagonist of LPA receptors significantly reduced the lymphatic vessel density in tumor and lymph node metastasis in tumor-bearing nude mice. Together, our results demonstrated that CRT is critical in PCa progression through the mediation of LPA-induced VEGF-C expression, implying that targeting the LPA signaling axis is a potential therapeutic strategy for PCa.     

Vascular Endothelial Growth Factor C-Induced Lymphangiogenesis Decreases Tumor Interstitial Fluid Pressure and Tumor

Characteristically, most solid tumors exhibit an increased tumor interstitial fluid pressure (TIFP) that directly contributes to the lowered uptake of macromolecular therapeutics into the tumor interstitium. Abnormalities in the tumor-associated lymph vessels are a central brick in the development and prolonged sustaining of an increased TIFP. In the current study, vascular endothelial growth factor C (VEGF-C) was used to enhance tumor-associated lymphangiogenesis as a new mechanism to actively reduce the TIFP by increased lymphatic drainage of the tumor tissue. Human A431 epidermoid vulva carcinoma cells were inoculated in NMRI nu/nu mice to generate a xenograft mouse model. Seven days after tumor cell injection, VEGF-C was peritumorally injected to induce lymphangiogenesis. Tumor growth and TIFP was lowered significantly over time in VEGF-C-treated tumors in comparison to control or VEGF-A-treated animals. These data demonstrate for the first time that actively induced lymphangiogenesis can lower the TIFP in a xenograft tumor model and apparently reduce tumor growth. This model represents a novel approach to modulate biomechanical properties of the tumor interstitium enabling a lowering of TIFP in vivo.     

Induction of tumor lymphangiogenesis by VEGF-C promotes breast cancer metastasis

Metastasis of breast cancer occurs primarily through the lymphatic system, and the extent of lymph node involvement is a key prognostic factor for the disease. Whereas the significance of angiogenesis for tumor progression has been well documented, the ability of tumor cells to induce the growth of lymphatic vessels (lymphangiogenesis) and the presence of intratumoral lymphatic vessels have been controversial. Using a novel marker for lymphatic endothelium, LYVE-1, we demonstrate here the occurrence of intratumoral lymphangiogenesis within human breast cancers after orthotopic transplantation onto nude mice. Vascular endothelial growth factor (VEGF)-C overexpression in breast cancer cells potently increased intratumoral lymphangiogenesis, resulting in significantly enhanced metastasis to regional lymph nodes and to lungs. The degree of tumor lymphangiogenesis was highly correlated with the extent of lymph node and lung metastases. These results establish the occurrence and biological significance of intratumoral lymphangiogenesis in breast cancer and identify VEGF-C as a molecular link between tumor lymphangiogenesis and metastasis.     

Role of macrophages in inflammatory lymphangiogenesis: Enhanced production of vascular endothelial growth factor C and D through NF-kappaB activation

The close association of inflammation, angiogenesis and cancer progression is now highlighted, and in this study we especially focused on a close association of inflammation and lymphangiogenesis. We found that proinflammatory cytokine, interleukin-1β (IL-1β), could induce lymphangiogenesis in mouse cornea through enhanced production of potent lymphangiogenic factors, VEGF-A, VEGF-C and VEGF-D. IL-1β-induced lymphangiogenesis, but not angiogenesis, was inhibited by administration of a selective anti-VEGF receptor-3 (VEGFR-3) neutralizing antibody. And in mouse cornea we observed recruitment of monocyte/macrophages and neutrophils by IL-1β implanted cornea. Depletion of macrophages by a bisphosphonate encapsulated in liposomes inhibited this IL-1β-induced lymphangiogenesis and also up-regulation of VEGF-A, VEGF-C, and VEGF-D. Furthermore, IL-1β-induced lymphangiogenesis and angiogenesis were suppressed by NF-κB inhibition with marked suppression of VEGF-A, VEGF-C, and VEGF-D expression.     

Expression of periostin in patients with non-small cell lung cancer: correlation with angiogenesis and lymphangiogenesis

The aim of this study was to evaluate periostin expression measured immunohistochemically in patients with non-small cell lung cancer (NSCLC) and to determine its association with clinical features, prognosis, angiogenesis, and lymphangiogenesis. We investigated periostin expression in a series of 88 patients with NSCLC. We also determined whether expression of periostin correlated with microvessel density and lymphatic microvessel density. Periostin was expressed in 42% of 88 patients. Its expression was significantly correlated with tumor size, lymph node metastasis, disease stage, and lymphatic invasion (p=0.0128, 0.0015, 0.0310 and 0.0273, respectively). There also was a significant relation between periostin expression and microvessel density and lymphatic microvessel density (all p<0.0001). Five-year survival rates were better in patients with negative periostin expression than in those with positive periostin expression (p=0.0044). Periostin expression was not significant in a multivariate additive model. Our findings show that periostin correlates with increased tumor progression and a worse prognosis in NSCLC, as well as with angiogenesis and lymphangiogenesis.     

卵巢上皮肿瘤淋巴转移与血管内皮生长因子C的表达

The aim of the present study was to explore the role of vascular endothelial growth factor-C (VEGF-C) in the process of angiogenesis, lymphangiogenesis and lymphatic metastasis in epithelial ovarian tumors. In situ hybridization and immunohistochemical staining for VEGF-C were performed in 30 epithelial ovarian carcinomas, 9 borderline tumors and 26 benign cystadenomas. Endothelial cells were immunostained with anti-VEGFR-3 pAb and anti-CD31 mAb, and VEGFR-3 positive vessels and microvessel density (MVD) were assessed by image analysis. VEGF-C mRNA and protein expression in ovarian epithelial carcinomas were significantly higher than that in borderline tumors and benign cystadenomas (p < 0.05 or p < 0.01). In ovarian epithelial carcinomas, VEGF-C protein expression, VEGFR-3 positive vessels and MVD were significantly higher in the cases of clinical stage III-IV and with lymphatic metastasis than those of clinical stage I-II and without lymphatic metastasis respectively (p < 0.05 or p < 0.01), VEGFR-3 positive vessels and MVD was significantly higher in the VEGF-C protein positive tumors than negative tumors (p < 0.05), VEGFR-3 positive vessels was significantly correlated with MVD(p < 0.01). These data suggest that VEGF-C might play a role in lymphatic metastasis via lymphangiogenesis and angiogenesis in epithelial ovarian carcinomas, and VEGF-C could be used as a biologic marker of metastasis in ovarian epithelial carcinomas.     

Lysophosphatidic acid upregulates vascular endothelial growth factor-C and tube formation in human endothelial cells through LPA(1/3), COX-2, and NF-kappaB activation- and EGFR transactivation-dependent mechanisms

Lysophosphatidic acid (LPA) is a lipid bioactive mediator which binds to G-protein-coupled receptors and activates a variety of cellular functions. LPA modulates multiple behaviors in endothelial cells, including cell proliferation and migration, capillary-like tube formation in vitro, activation of proteases, interactions with leukocytes, and expressions of inflammation-related genes, thereby regulating vessel formation. LPA has been reported to modulate the angiogenesis process. However, the role of LPA in the lymphangiogenesis process has not been studied. In this study, we showed that LPA upregulated vascular endothelial growth factor-C (VEGF-C) mRNA expression in human umbilical vein endothelial cells (HUVECs) and subsequent endothelial cell tube formation in vitro and in vivo. These enhancement effects were LPA(1)- and LPA(3)-dependent and required cyclooxygenase-2 (COX-2), endothelial growth factor receptor (EGFR) transactivation and activation of nuclear factor kappaB (NF-kappaB). Moreover, LPA induced the protein expressions of the lymphatic markers, Prox-1, LYVE-1, and podoplanin, in HUVECs, and these enhancement effects were dependent on LPA(1) and LPA(3) activation and EGFR transactivation. Our results demonstrated that LPA might regulate VEGF-C and lymphatic marker expression in endothelial cells, which contributes to endothelial cell tube formation in vitro and in vivo, thus facilitating endothelial cell participation in the lymphangiogenesis process. This study clarifies the signaling mechanism of LPA-regulated VEGF-C expression and lymphatic marker expressions in endothelial cells, which suggest that LPA may be a suitable target for generating therapeutics against lymphangiogenesis and tumor metastasis.