Hepatocyte growth factor promotes lymphatic vessel formation and function

The lymphatic vascular system plays a pivotal role in mediating tissue fluid homeostasis and cancer metastasis, but the molecular mechanisms that regulate its formation and function remain poorly characterized. A comparative analysis of the gene expression of purified lymphatic endothelial cells (LEC) versus blood vascular endothelial cells (BVEC) revealed that LEC express significantly higher levels of hepatocyte growth factor receptor (HGF-R). Whereas little or no HGF-R expression was detected by lymphatic vessels of normal tissues, HGF-R was strongly expressed by regenerating lymphatic endothelium during tissue repair and by activated lymphatic vessels in inflamed skin. Treatment of cultured LEC with HGF promoted LEC proliferation, migration and tube formation. HGF-induced proliferation of LEC did not require vascular endothelial growth factor receptor-3 activation, and HGF-induced cell migration was partially mediated via integrin alpha-9. Transgenic or subcutaneous delivery of HGF promoted lymphatic vessel formation in mice, whereas systemic blockade of HGF-R inhibited lymphatic function. These results identify HGF as a novel, potent lymphangiogenesis factor, and also indicate that HGF-R might serve as a new target for inhibiting pathological lymphangiogenesis.     

Blocking Fibroblast Growth Factor receptor signaling inhibits tumor growth, lymphangiogenesis, and metastasis

Fibroblast Growth Factor receptor (FGFR) activity plays crucial roles in tumor growth and patient survival. However, FGF (Fibroblast Growth Factor) signaling as a target for cancer therapy has been under-investigated compared to other receptor tyrosine kinases. Here, we studied the effect of FGFR signaling inhibition on tumor growth, metastasis and lymphangiogenesis by expressing a dominant negative FGFR (FGFR-2DN) in an orthotopic mouse mammary 66c14 carcinoma model. We show that FGFR-2DN-expressing 66c14 cells proliferate in vitro slower than controls. 66c14 tumor outgrowth and lung metastatic foci are reduced in mice implanted with FGFR-2DN-expressing cells, which also exhibited better overall survival. We found 66c14 cells in the lumen of tumor lymphatic vessels and in lymph nodes. FGFR-2DN-expressing tumors exhibited a decrease in VEGFR-3 (Vascular Endothelial Growth Factor Receptor-3) or podoplanin-positive lymphatic vessels, an increase in isolated intratumoral lymphatic endothelial cells and a reduction in VEGF-C (Vascular Endothelial Growth Factor-C) mRNA expression. FGFs may act in an autocrine manner as the inhibition of FGFR signaling in tumor cells suppresses VEGF-C expression in a COX-2 (cyclooxygenase-2) or HIF1-α (hypoxia-inducible factor-1 α) independent manner. FGFs may also act in a paracrine manner on tumor lymphatics by inducing expression of pro-lymphangiogenic molecules such as VEGFR-3, integrin α9, prox1 and netrin-1. Finally, in vitro lymphangiogenesis is impeded in the presence of FGFR-2DN 66c14 cells. These data confirm that both FGF and VEGF signaling are necessary for the maintenance of vascular morphogenesis and provide evidence that targeting FGFR signaling may be an interesting approach to inhibit tumor lymphangiogenesis and metastatic spread.     

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.     

Extracellular matrix regulates endothelial functions through interaction of VEGFR-3 and integrin alpha5beta1

Endothelium extracellular matrix (ECM) interactions can provide distinct spatial and molecular signals which control cellular proliferation, migration, and differentiation. Here, we investigated the role of fibronectin (FN), a major ECM protein, on the functions of lymphatic endothelial cells (LEC). We observed that FN, the ligand for integrin alpha5beta1, selectively promoted the growth of LEC as compared with vitronectin (VN) in the presence of the ligand for vascular endothelial growth factor receptor 3 [VEGFR-3 (VEGF-C156S)]. Upon investigating the mechanisms whereby ECM components regulate VEGFR-3 signaling, we found that FN transactivated VEGFR-3 and significantly enhanced the phosphorylation of VEGFR-3 induced by VEGF-C156S as compared to VN. An enhanced association of the integrin subunit alpha5 or beta1 with VEGFR-3, after stimulation with VEGF-C156S, was observed by co-immunoprecipitation. While blockade of integrin alpha5beta1 inhibited the VEGF-C156S-induced phosphorylation of VEGFR-3, no similar effect was obtained by blocking integrin alphavbeta3. FN also protected the endothelial cells from serum deprivation-induced apoptosis. Moreover, while the specific PI3 kinase inhibitor, LY294002, abolished this FN-mediated cell survival, the MAPK kinase inhibitor, PD98059, had no significant effect. Furthermore, a dominant-negative mutant of VEGFR-3 (G857R) reduced VEGF-C156S or FN-mediated cell survival, as well as the activities of PI3 kinase/Akt. Our results indicate that integrin alpha5beta1 participates in the activation of both VEGFR-3 and its downstream PI3 kinase/Akt signaling pathway, which is essential for FN-mediated lymphatic endothelial cell survival and proliferation.     

Isolated lymphatic endothelial cells transduce growth, survival and migratory signals via the VEGF-C/D receptor VEGFR-3

Vascular endothelial growth factor receptor-3 (VEGFR-3/Flt4) binds two known members of the VEGF ligand family, VEGF-C and VEGF-D, and has a critical function in the remodelling of the primary capillary vasculature of midgestation embryos. Later during development, VEGFR-3 regulates the growth and maintenance of the lymphatic vessels. In the present study, we have isolated and cultured stable lineages of blood vascular and lymphatic endothelial cells from human primary microvascular endothelium by using antibodies against the extracellular domain of VEGFR-3. We show that VEGFR-3 stimulation alone protects the lymphatic endothelial cells from serum deprivation-induced apoptosis and induces their growth and migration. At least some of these signals are transduced via a protein kinase C-dependent activation of the p42/p44 MAPK signalling cascade and via a wortmannin-sensitive induction of Akt phosphorylation. These results define the critical role of VEGF-C/VEGFR-3 signalling in the growth and survival of lymphatic endothelial cells. The culture of isolated lymphatic endothelial cells should now allow further studies of the molecular properties of these cells.     

Expression of vascular endothelial growth factor C in human pterygium

Vascular endothelial growth factor C (VEGF-C) and its receptor VEGFR-3 mediate lymphangiogenesis. In this study, we analyzed the expression of VEGF-C and VEGFR-3 as well as lymphatic vessels in the pterygium and normal conjunctiva of humans. Fifteen primary nasal pterygia and three normal bulbar conjunctivas, surgically removed, were examined in this study. The lymphatic vessel density (LVD) and blood vessel density were determined by the immunolabeling of D2-40 and CD31, markers for lymphatic and blood vessels, respectively. VEGF-C and VEGFR-3 expression in pterygial and conjunctival tissue proteins was detected by Western blotting and were evaluated using immunohistochemistry. The LVD was significantly higher in the pterygium than normal conjunctiva (p < 0.05). Western blot demonstrated high-level expression of VEGF-C and VEGFR-3 in the pterygium compared with normal conjunctiva. VEGF-C immunoreactivity was detected in the cytoplasm of pterygial and normal conjunctival epithelial cells. The number of VEGF-C-immunopositive cells in pterygial epithelial cells was significantly higher than in normal conjunctival cells (p < 0.05). VEGFR-3 immunoreactivity was localized in the D2-40-positive lymphatic endothelial cells. The present findings suggest the potential role of VEGF-C in the pathogenesis and development of a pterygium through lymphangiogenesis and the VEGF-C/VEGFR-3 pathway as a novel therapeutic target for the human pterygium.     

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.     

Inhibition of Lymphangiogenesis of Endothelial Progenitor Cells with VEGFR-3 siRNA Delivered with PEI-alginate Nanoparticles

Lymphangiogenesis is implicated in lymphatic metastasis of tumor cells. Recently, growing evidences show that endothelial progenitor cells (EPCs) are involved in lymphangiogenesis. This study has investigated effects of VEGF-C/VEGFR-3 (vascular endothelial growth factor receptor-3) signaling pathway on EPC differentiation and effectiveness of inhibiting lymphatic formation of EPCs with VEGFR-3 siRNA delivered in PEI (polyethylenimine)-alginate nanoparticles. CD34⁺VEGFR-3⁺ EPCs were sorted from mononuclear cells of human cord blood. Under induction with VEGF-C, the cells differentiated toward lymphatic endothelial cells. The nanoparticles were formulated with 25 kDa branched PEI and alginate. The size and surface charge of PEI-alginate nanoparticles loading VEGFR-3 siRNA (N/P = 16) are 139.1 nm and 7.56 mV respectively. VEGFR-3 siRNA specifically inhibited expression of VEGFR-3 mRNA in the cells. After treatment with PEI-alginate/siRNA nanocomplexes, EPCs could not differentiate into lymphatic endothelial cells, and proliferation, migration and lymphatic formation of EPC-derived cells were suppressed significantly. These results demonstrate that VEGFR-3 signaling plays an important role in differentiation of CD34⁺VEGFR-3⁺ EPCs. VEGFR-3 siRNA delivered with PEI-alginate nanoparticles can effectively inhibit differentiation and lymphangiogenesis of EPCs. Inhibiting VEGFR-3 signaling with siRNA/nanocomplexes would be a potential therapy for suppression of tumor lymphangiogenesis and lymphatic metastasis.     

Lymphatic endothelial cells, lymphangiogenesis, and extracellular matrix

Exciting studies involving the molecular regulation of lymphangiogenesis in lymphatic-associated disorders (e.g., wound healing, lymphedema and tumor metastasis) have focused renewed attention on the intrinsic relationship between lymphatic endothelial cells (LECs) and extracellular matrix (ECM) microenvironment. ECM molecules and remodeling events play a key role in regulating lymphangiogenesis, and the "functionality"-relating molecules, especially hyaluronan, integrins, reelin, IL-7, and matrix metalloproteinases, provide the most fundamental and critical prerequisite for LEC growth, migration, tube formation, and survival, although lymphangiogenesis is directly or/and indirectly controlled by VEGF-C/-D/VEGFR- 3- Prox-1-, Syk/SLP76-, podoplanin/Ang-2/Nrp-2-, FOXC2-, and other signaling pathways in embryonic and pathological processes. New knowledge regarding the differentiation of initial lymphatics should enable improvements in understanding of a variety of cytokines, chemokines, and other factors. The lymphatic colocalization with histochemical staining by using the novel molecular markers (e.g., LYVE-1), along with subsequent injection technique with ferritin or some tracer, will reveal functional and structural features of newly formed and preexisting lymphatics. Growing recognition of the multiple functions of ECM and LEC molecules for important physiological and pathological events may be helpful in identifying the crucial changes in tissues subjected to lymph circulation and ultimately in the search for rational therapeutic approaches to prevent lymphatic-associated disorders.     

Platelet activation receptor CLEC-2 regulates blood/lymphatic vessel separation by inhibiting proliferation, migration, and tube formation of lymphatic endothelial cells

The platelet activation receptor CLEC-2 plays crucial roles in thrombosis/hemostasis, tumor metastasis, and lymphangiogenesis, although its role in thrombosis/hemostasis remains controversial. An endogenous ligand for CLEC-2, podoplanin, is expressed in lymphatic endothelial cells (LECs). We and others have reported that CLEC-2-deficiency is lethal at mouse embryonic/neonatal stages associated with blood-filled lymphatics, indicating that CLEC-2 is essential for blood/lymphatic vessel separation. However, its mechanism, and whether CLEC-2 in platelets is necessary for this separation, remains unknown. We found that specific deletion of CLEC-2 from platelets leads to the misconnection of blood/lymphatic vessels. CLEC-2(+/+) platelets, but not by CLEC-2(-/-) platelets, inhibited LEC migration, proliferation, and tube formation but had no effect on human umbilical vein endothelial cells. Additionally, supernatants from activated platelets significantly inhibited these three functions in LECs, suggesting that released granule contents regulate blood/lymphatic vessel separation. Bone morphologic protein-9 (BMP-9), which we found to be present in platelets and released upon activation, appears to play a key role in regulating LEC functions. Only BMP-9 inhibited tube formation, although other releasates including transforming growth factor-β and platelet factor 4 inhibited proliferation and/or migration. We propose that platelets regulate blood/lymphatic vessel separation by inhibiting the proliferation, migration, and tube formation of LECs, mainly because of the release of BMP-9 upon activation by CLEC-2/podoplanin interaction.     

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.     

Mesenchymal stem cells promote lymphangiogenic properties of lymphatic endothelial cells

Lymphatic metastasis is one of the main prognostic factors concerning long‐term survival of cancer patients. In this regard, the molecular mechanisms of lymphangiogenesis are still rarely explored. Also, the interactions between stem cells and lymphatic endothelial cells (LEC) in humans have not been well examined. Therefore, the main objective of this study was to assess the interactions between mesenchymal stem cells (MSC) and LEC using in vitro angiogenesis assays. Juvenile LEC were stimulated with VEGF‐C, bFGF, MSC‐conditioned medium (MSC‐CM) or by co‐culture with MSC. LEC proliferation was assessed using a MTT assay. Migration of the cells was determined with a wound healing assay and a transmigration assay. To measure the formation of lymphatic sprouts, LEC spheroids were embedded in collagen or fibrin gels. The LEC's capacity to form capillary‐like structures was assessed by a tube formation assay on Matrigel^®. The proliferation, migration and tube formation of LEC could be significantly enhanced by MSC‐CM and by co‐culture with MSC. The effect of stimulation with MSC‐CM was stronger compared to stimulation with the growth factors VEGF‐C and bFGF in proliferation and transmigration assays. Sprouting was stimulated by VEGF‐C, bFGF and by MSC‐CM. With this study, we demonstrate the potent stimulating effect of the MSC secretome on proliferation, migration and tube formation of LEC. This indicates an important role of MSC in lymphangiogenesis in pathological as well as physiological processes.     

Bone Marrow-Derived Mesenchymal Stem Cells Drive Lymphangiogenesis

It is now well accepted that multipotent Bone-Marrow Mesenchymal Stem Cells (BM-MSC) contribute to cancer progression through several mechanisms including angiogenesis. However, their involvement during the lymphangiogenic process is poorly described. Using BM-MSC isolated from mice of two different backgrounds, we demonstrate a paracrine lymphangiogenic action of BM-MSC both in vivo and in vitro. Co-injection of BM-MSC and tumor cells in mice increased the in vivo tumor growth and intratumoral lymphatic vessel density. In addition, BM-MSC or their conditioned medium stimulated the recruitment of lymphatic vessels in vivo in an ear sponge assay, and ex vivo in the lymphatic ring assay (LRA). In vitro, MSC conditioned medium also increased the proliferation rate and the migration of both primary lymphatic endothelial cells (LEC) and an immortalized lymphatic endothelial cell line. Mechanistically, these pro-lymphangiogenic effects relied on the secretion of Vascular Endothelial Growth Factor (VEGF)-A by BM-MSC that activates VEGF Receptor (VEGFR)-2 pathway on LEC. Indeed, the trapping of VEGF-A in MSC conditioned medium by soluble VEGF Receptors (sVEGFR)-1, -2 or the inhibition of VEGFR-2 activity by a specific inhibitor (ZM 323881) both decreased LEC proliferation, migration and the phosphorylation of their main downstream target ERK1/2. This study provides direct unprecedented evidence for a paracrine lymphangiogenic action of BM-MSC via the production of VEGF-A which acts on LEC VEGFR-2.     

Regulation of lymphatic capillary regeneration by interstitial flow in skin

Decreased interstitial flow (IF) in secondary lymphedema is coincident with poor physiological lymphatic regeneration. However, both the existence and direction of causality between IF and lymphangiogenesis remain unclear. This is primarily because the role of IF and its importance relative to the action of the prolymphangiogenic growth factor vascular endothelial growth factor (VEGF)-C (which signals primarily through its receptor VEGFR-3) are poorly understood. To clarify this, we explored the cooperative roles of VEGFR-3 and IF in a mouse model of lymphangiogenesis in regenerating skin. Specifically, a region of lymphangiogenesis was created by substituting a portion of mouse tail skin with a collagen gel within which lymphatic capillaries completely regenerate over a period of 60 days. The relative importance of IF and VEGF-C signaling were evaluated by either inhibiting VEGFR-3 signaling with antagonistic antibodies or by reducing IF. In some cases, VEGF-C signaling was then increased with exogenous protein. To clarify the role of IF, the distribution of endogenous matrix metalloproteinases (MMPs) and VEGF-C within the regenerating region was determined. It was found that inhibition of either VEGFR-3 or IF suppressed endogenous lymphangiogenesis. Reduction of IF was found to decrease lymphatic migration and transport of endogenous MMP and VEGF-C through the regenerating region. Therapeutic VEGF-C administration restored lymphangiogenesis following inhibition of VEGFR-3 but did not increase lymphangiogenesis following inhibition of IF. These results identify IF as an important regulator of the pro-lymphangiogenic action of VEGF-C.     

Vascular endothelial growth factor (VEGF) receptor-2 signaling mediates VEGF-C(deltaNdeltaC)- and VEGF-A-induced angiogenesis in vitro

Angiogenesis and lymphangiogenesis are regulated by members of the vascular endothelial growth factor (VEGF) family of cytokines, which mediate their effects via tyrosine kinase VEGF receptors -1, -2, and -3. We have used wild-type and mutant forms of VEGFs -A, -B, and -C, a pan-VEGFR tyrosine kinase inhibitor (SU5416) as well as neutralizing anti-VEGFR-2 antibodies, to determine which VEGF receptor(s) are required for bovine endothelial cell invasion and tube formation in vitro. This was compared to the ability of these cytokines to induce expression of members of the plasminogen activator (PA)-plasmin system. We found that cytokines which bind VEGFR-2 (human VEGF-A, human VFM23A, human VEGF-C(deltaNdeltaC), and rat VEGF-C(152)) induced invasion, tube formation, urokinase-type-PA, tissue-type-PA, and PA inhibitor-1, invasion and tube formation as well as signaling via the MAP kinase pathway were efficiently blocked by SU5416 and anti-VEGFR-2 antibodies. In contrast, cytokines and mutants which exclusively bind VEGFR-1 (human VFM17 and human VEGF-B) had no effect on invasion and tube formation or on the regulation of gene expression. We were unable to identify cytokines which selectively stimulate bovine VEGFR-3 in our system. Taken together, these findings point to the central role of VEGFR-2 in the angiogenic signaling pathways induced by VEGF-C(deltaNdeltaC) and VEGF-A.     

Role of lymphangiogenic factors in tumor metastasis

Nearly four centuries after the discovery of lymphatic vessels, the molecular mechanisms underlying their development are beginning to be elucidated. Vascular endothelial growth factor C (VEGF-C) and VEGF-D, via signaling through VEGFR-3, appear to be essential for lymphatic vessel growth. Observations from clinicopathological studies have suggested that lymphatic vessels serve as the primary route for the metastatic spread of tumor cells to regional lymph nodes. Recent studies in animal models have provided convincing evidence that tumor lymphangiogenesis facilitates lymphatic metastasis. However, it is not clear how tumor-associated lymphangiogenesis is regulated, and little is known about how tumor cells escape from the primary tumor and gain entry into the lymphatics. This review examines some of these issues and provides a brief summary of the recent developments in this field of research.     

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.     

Expression of vascular endothelial growth factors VEGF- C and D, VEGFR-3, and comparison of lymphatic vessels density labeled with D2-40 antibodies as a prognostic factors in vulvar intraepithelial neoplasia (VIN) and invasive vulvar cancer

OBJECTIVE: The aim of this study was to compare the immunohistochemical expression of vascular endothelial growth factors VEGF-C and D, as well as the expression of VEGFR-3 in VIN and vulvar invasive cancer and to compare the density of lymphatic marker D2-40 antibody in both groups, and to compare them with different clinicopathologic features. Materials & Methods: The study was performed using tissue material and clinical data from 100 women diagnosed with VIN and 100 women diagnosed with invasive vulvar cancer. Results: No significant differences were found in the expression of VEGF-C and -D or VEGFR-3 between those patients with VIN and those with invasive vulvar cancers. Weak expression of VEGF-C was confirmed only in two cases of the analyzed series; in all cases, expression of VEGF-D and VEGFR-3 was observed. The strongest expression of VEGF-D and VEGFR-3 was observed in the group of invasive cancers. The highest density of lymphatic vessels per 2 mm was observed in VIN. In the cancer group, small lymphatic vessels with a narrow oval lumen were observed. Moreover, in two cases of vulvar cancer, the presence of intratumoral lymphatic vessels was observed. Conclusions: These results suggest that lymphangiogenesis begins at the preinvasive stage of vulvar carcinogenesis and suggests the important role of VEGF-C, VEGF-D, VEGFR-3 and LV (D2-40) as prognostic factors in the process of carcinogenesis in the vulvar area.