Whole-body imaging of lymphovascular niches identifies pre-metastatic roles of midkine.

Cutaneous melanoma is a type of cancer with an inherent potential for lymph node colonization, which is generally preceded by neolymphangiogenesis. However, sentinel lymph node removal does not necessarily extend the overall survival of patients with melanoma. Moreover, lymphatic vessels collapse and become dysfunctional as melanomas progress. Therefore, it is unclear whether (and how) lymphangiogenesis contributes to visceral metastasis. Soluble and vesicle-associated proteins secreted by tumours and/or their stroma have been proposed to condition pre-metastatic sites in patients with melanoma. Still, the identities and prognostic value of lymphangiogenic mediators remain unclear. Moreover, our understanding of lymphangiogenesis (in melanomas and other tumour types) is limited by the paucity of mouse models for live imaging of distal pre-metastatic niches. Injectable lymphatic tracers have been developed, but their limited diffusion precludes whole-body imaging at visceral sites. Vascular endothelial growth factor receptor 3 (VEGFR3) is an attractive 'lymphoreporter' because its expression is strongly downregulated in normal adult lymphatic endothelial cells, but is activated in pathological situations such as inflammation and cancer. Here, we exploit this inducibility of VEGFR3 to engineer mouse melanoma models for whole-body imaging of metastasis generated by human cells, clinical biopsies or endogenously deregulated oncogenic pathways. This strategy revealed early induction of distal pre-metastatic niches uncoupled from lymphangiogenesis at primary lesions. Analyses of the melanoma secretome and validation in clinical specimens showed that the heparin-binding factor midkine is a systemic inducer of neo-lymphangiogenesis that defines patient prognosis. This role of midkine was linked to a paracrine activation of the mTOR pathway in lymphatic endothelial cells. These data support the use of VEGFR3 reporter mice as a 'MetAlert' discovery platform for drivers and inhibitors of metastasis.

Ascending Vasa Recta Are Angiopoietin/Tie2-Dependent Lymphatic-Like Vessels.

Urinary concentrating ability is central to mammalian water balance and depends on a medullary osmotic gradient generated by a countercurrent multiplication mechanism. Medullary hyperosmolarity is protected from washout by countercurrent exchange and efficient removal of interstitial fluid resorbed from the loop of Henle and collecting ducts. In most tissues, lymphatic vessels drain excess interstitial fluid back to the venous circulation. However, the renal medulla is devoid of classic lymphatics. Studies have suggested that the fenestrated ascending vasa recta (AVRs) drain the interstitial fluid in this location, but this function has not been conclusively shown. We report that late gestational deletion of the angiopoietin receptor endothelial tyrosine kinase 2 (Tie2) or both angiopoietin-1 and angiopoietin-2 prevents AVR formation in mice. The absence of AVR associated with rapid accumulation of fluid and cysts in the medullary interstitium, loss of medullary vascular bundles, and decreased urine concentrating ability. In transgenic reporter mice with normal angiopoietin-Tie2 signaling, medullary AVR exhibited an unusual hybrid endothelial phenotype, expressing lymphatic markers (prospero homeobox protein 1 and vascular endothelial growth factor receptor 3) as well as blood endothelial markers (CD34, endomucin, platelet endothelial cell adhesion molecule 1, and plasmalemmal vesicle-associated protein). Taken together, our data redefine the AVRs as Tie2 signaling-dependent specialized hybrid vessels and provide genetic evidence of the critical role of AVR in the countercurrent exchange mechanism and the structural integrity of the renal medulla.

Selective Stimulation of Cardiac Lymphangiogenesis Reduces Myocardial Edema and Fibrosis Leading to Improved Cardiac Function Following Myocardial Infarction.

BACKGROUND: The lymphatic system regulates interstitial tissue fluid balance, and lymphatic malfunction causes edema. The heart has an extensive lymphatic network displaying a dynamic range of lymph flow in physiology. Myocardial edema occurs in many cardiovascular diseases, eg, myocardial infarction (MI) and chronic heart failure, suggesting that cardiac lymphatic transport may be insufficient in pathology. Here, we investigate in rats the impact of MI and subsequent chronic heart failure on the cardiac lymphatic network. Further, we evaluate for the first time the functional effects of selective therapeutic stimulation of cardiac lymphangiogenesis post-MI. METHODS AND RESULTS: We investigated cardiac lymphatic structure and function in rats with MI induced by either temporary occlusion (n=160) or permanent ligation (n=100) of the left coronary artery. Although MI induced robust, intramyocardial capillary lymphangiogenesis, adverse remodeling of epicardial precollector and collector lymphatics occurred, leading to reduced cardiac lymphatic transport capacity. Consequently, myocardial edema persisted for several months post-MI, extending from the infarct to noninfarcted myocardium. Intramyocardial-targeted delivery of the vascular endothelial growth factor receptor 3-selective designer protein VEGF-CC152S, using albumin-alginate microparticles, accelerated cardiac lymphangiogenesis in a dose-dependent manner and limited precollector remodeling post-MI. As a result, myocardial fluid balance was improved, and cardiac inflammation, fibrosis, and dysfunction were attenuated. CONCLUSIONS: We show that, despite the endogenous cardiac lymphangiogenic response post-MI, the remodeling and dysfunction of collecting ducts contribute to the development of chronic myocardial edema and inflammation-aggravating cardiac fibrosis and dysfunction. Moreover, our data reveal that therapeutic lymphangiogenesis may be a promising new approach for the treatment of cardiovascular diseases.

[Epithelioid sarcoma with mesothelial and lymphatic endothelial differentiation: a clinicopathologic analysis of 10 cases].

Objective: To investigate the multidirectional differentiation potential in epithelioid sarcoma (ES), with special emphasis on its mesothelial and lymphatic endothelial markers expression. Methods: Ten cases of distal-type ES were included. The clinical, histological, and immunohistochemical(including mesothelial and lymphatic endothelial markers expression)features and follow-up data were evaluated. Results: The patients aged between 8 to 66 years. Five cases were male and five were female. The tumors were located at the palm (2 cases), wrist (3 cases), upper arm (2 cases), poplitealfossa (1 case), lower leg (1 case) and thigh (1 case), respectively. Clinically, most cases presented as painful, firm subcutaneous nodules. Histologically, the tumors were mainly composed of epithelioid, rhabdoid, spindle, or transitional cells, with abundant eosinophilic cytoplasm, oval and vesicular nucleus, and one or more prominent nucleoli. They were arranged in nodular, diffuse nodular or sheet like growth patterns, frequently with necrosis at the center with vague granulomatous configuration. Immunohistochemically, all tumors expressed cytokeratins, epithelial membrane antigen, CD34, desmin, mesothelial markers such as Calretinin, WT1, D2-40, M2A, vascular and lymphatic endothelial markers FLI-1, and VEGFR-3. The tumor cells did not express CD31, FⅧRAg, HHF35, HMB45, Melan A, MyoD1, myogenin, S-100 protein and SMA. All 10 patients underwent radical resection or extended excision, with additional radiotherapy or chemotherapy. During the follow-up from October 2012 to August 2016, seven cases showed recurrences and metastases within 2 months to 2 years. Five patients died of the disease due to widespread metastases. Conclusions: ES may show a wide spectrum of morphology, and display a multidirectional differentiating capabilities including towards mesothelial and lymphatic endothelial cells. As such, its diagnosis and differential diagnosis are particularly important as it is easily confused with other tumors with similar morphology or immune phenotype.

Interleukin 12 shows a better curative effect on lung cancer than paclitaxel and cisplatin doublet chemotherapy.

BACKGROUND: Interleukin 12 (IL-12) is a cytokine that has been reported to exhibit potent tumoricidal effects in animal tumor models. A combined approach using Paclitaxel and platinum-based doublet chemotherapy is the most commonly used backbone regimen for treating lung cancer. Despite numerous studies regarding the anti-tumor effects of IL-12 and the widespread use of conventional chemotherapy, few direct comparisons of IL-12 and conventional chemotherapy in the treatment of lung cancer have been performed. METHODS: We compared IL-12 to paclitaxel and cisplatin doublet chemotherapy in terms of efficacy against lung cancer in mouse models. The antitumor effect was measured by survival assays, histological analyses and imaging analyses. The cytokine levels were assessed using enzyme linked immunosorbent assay (ELISA) and flow cytometry (FACS). The spleen sizes were measured. CD31, CD105 and Vascular endothelial growth factor receptor 3 (VEGFR3) were analyzed using immunofluorescence. Matrix metalloprotein-9 (MMP-9) and cadherin 1 (CDH1) transcript levels were measured by quantitative PCR. Tumor cells apoptosis were examined by Tunel assay. RESULTS: The results showed that IL-12 treatment inhibited lung tumor growth, resulting in the long-term survival of lung cancer-bearing mice. Further examination revealed that IL-12 rapidly activated NK cells to secrete IFN-γ, resulting in the inhibition of tumor angiogenesis. In contrast, paclitaxel and cisplatin doublet chemotherapy did not show the expected efficacy in orthotopic lung cancer models; the IFN-γ levels were not increased after this treatment, and the number of peripheral lymphocytes was reduced. CONCLUSION: Together, these animal model data indicate that IL-12 shows a better curative effect than PTX + CDDP doublet chemotherapy.

VEGF receptor subtypes may serve as novel prognostic factors and putative indicators for anti VEGF receptor treatment response in renal cell carcinoma cases.

PURPOSE: Targeted therapies are novel treatment options for renal cell carcinoma (RCC). Of the target molecules investigated, vascular endothelial growth factor receptors (VEFGRs) were seldom evaluated. The current study investigated the prognostic significance of VEGFRs and IMP-3 as a potential prognostic markers. METHODS: Pathological material and clinical files of 100 patients with RCC were retrospectively evaluated. For each case, the clinical outcome and disease stage were assessed and resected materials were histologically reevaluated. VEGFR-2, VEGFR-3 and IMP-3 expression of tumor samples were analyzed with immunohistochemistry. These expressions were compared with prognosis and clinicopathological variables. RESULTS: Five-year overall survival (OS) was 80% in the whole cohort. Mean survival was 20.3±1.9 months in metastatic disease (95%CI:16.4-24.2). Two-year OS was 20% and 5-year OS was zero in the metastatic group. Survival was significantly longer in VEGFR-2 expressing group than in the nonexpressing group (78.7±2.6 vs 63.9±6; 95%CI:73.7-84 and 52.1-75.7, respectively; p=0.031). VEGFR-3 and IMP-3 expressions were not significantly correlated with survival. In the non-metastatic group mean OS was 82.6±2.1 months and 2- and 5-year OS were 96 and 88%, respectively. CONCLUSIONS: Since VEGFRs were expressed on all histological subtypes and significantly correlated with survival, assessment of VEGFR-2 and VEGFR-3 on tumor samples might serve as a putative prognostic factor in RCC cases. These expressions might identify a subset of patients that may benefit from antiangiogenic treatments targeting VEGFR receptors.

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.

Papillary thyroid carcinoma involving cervical neck lymph nodes: correlations with lymphangiogenesis and ultrasound features.

Stratification of risk factors for cervical lymph node metastasis (LNM) in thyroid papillary carcinoma is important for providing standards for post-operative adjuvant radio-iodine therapy and for patient prognosis. We investigated pathological factors based on the lymphatic vessel system and radiological features associated with tumor with cervical neck LNM. Among patients who had undergone thyroidectomy confirmed to be papillary thyroid carcinoma, we selected 126 age-sex matched paired patients without cervical LNM (group 1) and with LNM (group 2) to evaluate risk factors. Pathological factors evaluated were size, multiplicity, and extra thyroid extension state, based on the pathological reports using stored data. The lymphatic vessel density (LVD) of each tumor was evaluated by staining for VEGFR-3 and D2-40 and correlated with cervical LNM state. Malignant ultrasound features were evaluated to compare the differences between these two groups. Larger tumor size, multiplicity, extrathyroid extension were more common in group 2 (p<0.05). The median percentage of VEGFR-3 for group 1 was 20 (range 0-30) and D2-40 was 13 (range 7-23) while for group 2, VEGFR-3 was 80 (70-90) and D2-40 was 78 (54-114). LVD measured by intratumoral D2-40 staining was 20.6% and 79.4% for group 1 and group 2, respectively. Intra-tumoral lymphatics measured by D2-40 stain had a strong correlation with cervical LNM (Odds 1.230, CI 1.01.-1.499 p value 0.040). Ultrasound (US) features had no significant differences between the two groups although calcifications tended to be higher in group 2 (84% vs. 76% p=0.264). Lymphatic vessel density and nodule echogenicity were not associated with LNM. Intratumoral lymphangiogenesis was most strongly associated with LNM and thus, could be a useful predictive marker for cervical LNM.

Vascular endothelial growth factors and their receptors and regulators in gestational trophoblastic diseases and normal placenta.

OBJECTIVE: To study the expression of vascular endothelial growth factors (VEGFs), placental growth factor (PLGF) and their receptors (VEGFR-1, -2, -3) and their regulators (IL-6, CD147) in normal placenta and gestational trophoblastic disease (GTD) in order to evaluate their potential role in the biology of GTD. STUDY DESIGN: Paraffin sections of 10 normal, first-trimester placentas, 10 partial moles, 10 complete moles, 5 choriocarcinomas and 5 placental site trophoblastic tumors (PSTTs) were studied immunohistochemically for expression of VEGFR-1, VEGFR-2, VEGFR-3, IL-6, PLGF and CD147. Immunolocalization of VEGF, Angiopoietin-1 and Angiopoietin-2 was performed on 5 choriocarcinomas and 5 PSTTs. The levels of VEGF and VEGFR-2 were determined in supernatants and lysates of normal trophoblast, JEG-3 and JAR choriocarcinoma cells with electrochemiluminescence assays. RESULTS: The normal placenta had significantly stronger expression of VEGFR-2 than did those of partial and complete mole (p = 0.001, p = 0.003). VEGF, Angiopoietin-1 and Angiopoietin-2 expression in PSTT were significantly higher than those in choriocarcinoma (p = 0.002, p= 0.01, p = 0.038). Choriocarcinoma showed stronger intensity of staining for VEGFR-3 than did normal placenta, partial and complete mole (p = 0.036, p = 0.038, p = 0.05). Choriocarcinoma had significantly stronger staining of CD147 than did partial and complete mole (p<0.01, p<0.01). PSTT exhibited significantly stronger staining for IL-6 than did choriocarcinoma (p = 0.03). CONCLUSION: PSTTs exhibited strong staining for VEGF, and choriocarcinoma showed strong staining for VEGFR-3. Agents that inhibit the activity of VEGF and VEGF receptors may prove to be useful in the therapy of gestational trophoblastic neoplasia.

Detecting host factors involved in virus infection by observing the clustering of infected cells in siRNA screening images.

MOTIVATION: Detecting human proteins that are involved in virus entry and replication is facilitated by modern high-throughput RNAi screening technology. However, hit lists from different laboratories have shown only little consistency. This may be caused by not only experimental discrepancies, but also not fully explored possibilities of the data analysis. We wanted to improve reliability of such screens by combining a population analysis of infected cells with an established dye intensity readout. RESULTS: Viral infection is mainly spread by cell-cell contacts and clustering of infected cells can be observed during spreading of the infection in situ and in vivo. We employed this clustering feature to define knockdowns which harm viral infection efficiency of human Hepatitis C Virus. Images of knocked down cells for 719 human kinase genes were analyzed with an established point pattern analysis method (Ripley's K-function) to detect knockdowns in which virally infected cells did not show any clustering and therefore were hindered to spread their infection to their neighboring cells. The results were compared with a statistical analysis using a common intensity readout of the GFP-expressing viruses and a luciferase-based secondary screen yielding five promising host factors which may suit as potential targets for drug therapy. CONCLUSION: We report of an alternative method for high-throughput imaging methods to detect host factors being relevant for the infection efficiency of viruses. The method is generic and has the potential to be used for a large variety of different viruses and treatments being screened by imaging techniques.

Vascular endothelial growth factor receptor 2 (VEGFR-2) signalling activity in paediatric pilocytic astrocytoma is restricted to tumour endothelial cells.

AIMS: Tumours depend on angiogenesis for enhanced tumour cell survival and progression. Vascular endothelial growth factor receptor (VEGFR) signalling plays a major part in this process. Previously, we evaluated tyrosine kinase activity in paediatric brain tumour tissue lysates using a peptide microarray containing 144 different tyrosine kinase peptide substrates. When applied to paediatric pilocytic astrocytoma tissue, this analysis revealed extensive phosphorylation of VEGFR-derived peptides. The aim of the current study was to validate this result and determine the presence of VEGFR-2 activity in paediatric pilocytic astrocytoma as the main VEGFR in terms of mitogenic signalling. In addition, the localization of VEGFR1-3 mRNA expression was assessed. METHODS: VEGFR-2 phosphorylation was determined by adopting a proximity ligation assay approach. Enrichment of endothelial markers and VEGFRs in tumour endothelium was determined by quantitative polymerase chain reaction (qPCR) analysis of laser-microdissected blood vessels. RESULTS: Proximity ligation assays on tumour cryosections showed the presence of phosphorylation of VEGFR-2, which primarily localized to vascular endothelium. qPCR analysis of endothelial markers and VEGFRs showed a 13.6-fold average enrichment of VEGFR-2 expression in the laser-microdissected endothelium compared to whole tumour. Also the expression of VEGFR-1 and -3 was highly enriched in the endothelium fraction with an average fold-enrichment of 16.5 and 50.8 respectively. CONCLUSIONS: Phosphorylated VEGFR-2 is detected on endothelial cells in paediatric pilocytic astrocytoma. Furthermore, endothelial cells are the main source of VEGFR1-3 mRNA expression. This suggests a crucial role for VEGF/VEGFR-induced angiogenesis in the progression and maintenance of these tumours.

Suppression of vascular endothelial growth factor receptor 3 (VEGFR3) and vascular endothelial growth factor C (VEGFC) inhibits hypoxia-induced lymph node metastases in cervix cancer.

OBJECTIVES: We have examined the role of VEGFC/VEGFR3 signaling in lymph node metastasis and growth of orthotopic human ME180 and SiHa cervical xenograft models following exposure to hypoxia. Our previous studies showed that growth of these tumors under conditions of cyclic hypoxia increased nodal metastasis. METHODS: Mice bearing orthotopic tumors were subjected to cyclic hypoxia at 7% O(2)/air 10min cycles 4h/day/2weeks. Knockdown of vegfc was carried out by shRNA and inhibition of VEGFR3 was conducted by blocking antibodies for the mouse and human proteins. VEGFR3 protein expression was detected by Western blotting. Immunohistochemical staining was used to assess CA9, CD31, LYVE1 and Ki67 labeling. Gene expression was determined by real time PCR. RESULTS: Knock down of vegfc or inhibition of VEGFR3 with blocking antibody reduced metastases under normoxic and cyclic hypoxia conditions. A reduction in lymphatics and blood vessel formation and a decrease in tumor cell proliferation was observed following vegfc knockdown and VEGFR3 inhibition. VEGFR3 expression was upregulated at the mRNA and protein levels following hypoxia. CONCLUSIONS: Collectively, our results indicate that anti-VEGFR3 antibody inhibits vegfc-induced tumor lymphangiogenesis and metastasis and that vegfc knockdown in the tumor cells causes a similar inhibitory effect on lymph node metastasis. These results suggest that the effects of vegfc/VEGFR3 on the progression of tumor cells to form lymph node metastases occur primarily under an hypoxic tumor microenvironment.

Myopericytoma arising from myopericytosis-a hitherto unrecognized entity within the lung.

Two cases of myopericytosis combined with pericytoma originating within the lung are reported. These are rare pulmonary tumors. The differential diagnosis for hemangiopericytoma and pericytic tumors with glomus elements is discussed. Both myopericytic lesions mimic other lesions, which are more commonly seen in the lung. Based on the expression of vascular growth factor receptors 2 and 3, an antiangiogenic therapy was suggested for the patient with the myopericytoma. A treatment with an angiogenesis inhibitor resulted in a regression of the tumor, but not the precursor lesion. Probably a more specific therapy using tyrosine kinase inhibitors for VEGFR2/3 might better control these myopericytic proliferations.

Significance of phospho-vascular endothelial growth factor receptor-2 expression in pancreatic cancer.

Vascular endothelial growth factor receptors (VEGFRs) are mainly expressed by endothelial cells, but they are also expressed by some cancer cells, including pancreatic cancer. The objective of this study was to evaluate the significance of VEGFRs expression in pancreatic cancer cells. A total of 107 primary pancreatic tumors were stained with antibodies against VEGFR-1, VEGFR-2, phospho-VEGFR-2 (pVEGFR-2), VEGFR-3, VEGF-A, VEGF-C, and VEGF-D. VEGFR-2 and pVEGFR-2 expression were positive in 74 (69%) and 54 (50%) of 107 pancreatic cancers. There was a significant correlation (P < 0.001) between VEGFR-2 expression and pVEGFR-2 expression. pVEGFR-2 was significantly associated with invasion to the anterior capsule of pancreas (P = 0.032) and arterial invasion (P = 0.012). In contrast, VEGFR-1 and VEGFR-3 expression was only observed in 13 (12%) and 15 (14%) of 107 pancreatic cancers, and was not associated with any clinicopathological features. The prognosis of pVEGFR-2 positive patients with stage IIA tumors was significantly (P = 0.0441) poorer than that of pVEGFR-2-negative patients. VEGF-A, VEGF-C, and VEGF-D expression was positive in 42 (39%), 82 (77%), and 39 (36%) of 107 pancreatic cancers, respectively. The prognosis for VEGF-A-positive patients was significantly (P = 0.0425) poor, but not for VEGF-C-positive and VEGF-D-positive patients. A multivariate analysis indicated pVEGFR-2 expression to be an independent prognostic factor, but not VEGF-A. These findings suggested that VEGFR-2 signaling might therefore be associated with the prognosis of patients with pancreatic cancer. The expression of pVEGFR-2 might be a novel predictive prognostic marker for patients with pancreatic cancers, especially at clinical stage IIA.

Potential role for vascular endothelial growth factor-D as an autocrine factor for human gastric carcinoma cells.

Vascular endothelial growth factor (VEGF)-D induces lymphangiogenesis by activating VEGF receptor (VEGFR)-3, which is expressed mainly by lymphatic endothelial cells. VEGFR-3 has also been detected in several types of malignant cells, but the significance of VEGFR-3 expression by malignant cells remains unclear. We examined the expression and function of VEGF-D/VEGFR-3 in human gastric carcinoma cells. Expression of VEGF-D and VEGFR-3 was analyzed in three human gastric carcinoma cell lines and 29 surgical specimens. cDNA microarray analysis was used to examine the effect of VEGF-D on the expression of genes associated with disease progression in VEGFR-3-expressing KKLS cells. VEGF-D-transfected cells and control cells were transplanted into the gastric wall of nude mice. In 10 of the 29 (34%) gastric carcinoma specimens and two of the three cell lines, cancer cells expressed both VEGF-D and VEGFR-3. In vitro treatment of KKLS cells with exogenous VEGF-D increased expression of cyclin D1 and Bcl-2 and stimulated cell proliferation. VEGF-D transfection into KKLS cells resulted in stimulation of angiogenesis, lymphangiogenesis, and cell proliferation, and in inhibition of apoptosis. VEGF-D may participate in the progression of human gastric carcinoma by acting via autocrine and paracrine mechanisms.

Different significance between intratumoral and peritumoral lymphatic vessel density in gastric cancer: a retrospective study of 123 cases.

BACKGROUND: Patients with gastric cancer in China have worse outcome and poorer prognosis. Tumor-induced lymphangiogenesis plays a crucial role in metastasis and tumor progression. The intratumoral and peritumoral lymphatics were supposed to have different biological effects. Three major growth factors, vascular endothelial growth factor- (VEGF)-A, VEGF-C and VEGF-D, are involved in the activation process via their receptors (VEGFRs). The purpose of current study is to investigate the significant difference between intratumoral and peritumoral lymphatic vessel density (LVD) in gastric cancer and their correlations with lymphangiogenetic growth factors. METHODS: Intratumoral LVD (I-LVD) and peritumoral LVD (P-LVD) of 123 patients with primary gastric cancer were assessed after staining with D2-40, and confirmed by double staining with D2-40/CD34. Proliferative activity of lymphatics endothelium was evaluated by double staining with D2-40/Ki-67. The associations were analyzed between I-LVD/P-LVD and the expression level of VEGF-A, VEGF-C, VEGF-D and the receptor VEGFR-3, which was measured by immunohistochemistry (IHC). The correlations of I-LVD and P-LVD with patient prognosis were also valued. RESULTS: (1) The peritumoral lymphatics (PTLs) were relatively enlarged with dilated lumen compared with the intratumoral lymphatics (ITLs). Increased P-LVD was significantly higher than I-LVD (P < 0.05). (2) P-LVD was found significantly associated with lymph node metastasis (LNM) (P < 0.001), lymphatic vessel invasion (LVI) (P < 0.001), VEGF-C (P = 0.003), VEGF-D expression level (P = 0.005) and VEGFR-3 expression level (P < 0.001) in peritumoral tissues, despite no significant association was found between above variants with I-LVD. However, increased I-LVD was demonstrated to be associated with decreased tumor volume (P < 0.001). Neither I-LVD nor P-LVD was correlated with VEGF-A expression (P > 0.05). (3) Proliferative activity of lymphatics endothelium was observed in PTLs, in spite of ITLs. (4) Increased P-LVD, but not I-LVD, was indicated to be an independent risk factor for lymph node metastasis by multivariate logistic regression analysis, and was related to worse disease-free survival and overall survival. CONCLUSIONS: PTLs play roles in gastric cancer progression. Increased P-LVD, but not I-LVD, was significantly associated with VEGF-C/-D/VEGFR-3 system, and could be an independent risk factor for lymph node metastasis and a prognostic factor in gastric cancer.

Vascular endothelial growth factor-C promotes the growth and invasion of gallbladder cancer via an autocrine mechanism.

Vascular endothelial growth factor-C (VEGF-C) has a well-defined action on neoplastic lymphangiogenesis and angiogenesis through VEGF receptor-3 (VEGFR-3) and VEGFR-2, respectively, which are generally expressed in endothelial cells. The function of the VEGF-C/receptors pathway in tumor cell types is largely unknown. In this study, we examined the expression and role of VEGF-C/receptors in gallbladder cancer (GBC) cells. We examined the expression of VEGF-C in 50 surgical specimens from gallbladder cancer and three human gallbladder cancer cell lines. Both siRNA and neutralizing antibody to deplete the expression of VEGF-C were used to characterize the biological effect of VEGF-C in GBC NOZ cells. Furthermore, we examined the expression of its receptors, VEGFR-3 and VEGFR-2, in three human GBC cell lines. Our results are as follows: The expression of VEGF-C in the invasive marginal portion was significantly higher than the expression in the central portions. All the three GBC cell lines expressed VEGF-C. Treatment of NOZ cells with VEGF-C siRNA or a neutralizing antibody suppressed cell proliferation and invasion. Moreover, all the three GBC cell lines expressed VEGFR3, but only the NOZ cells expressed VEGFR-2 mRNA. Treatment of NOZ cells with a VEGFR-3 neutralizing antibody suppressed cell invasion, but treatment of NOZ cells with a VEGFR-2 neutralizing antibody suppressed cell proliferation and invasion. In conclusion, GBC cells express both VEGF-C and its receptors. VEGF-C may have a role in the progressive growth and invasion of human GBC through an autocrine mechanism.

Absence of lymphatic vessels in human dental pulp: a morphological study.

Few and controversial data are available in the literature regarding the presence of lymphatic vessels in the human dental pulp. The present study was designed to examine morphologically the existence of a lymph drainage system in human dental pulp. Human dental pulp and skin sections were immunohistochemically stained with specific antibodies for lymphatic endothelium (D2-40, LYVE-1, VEGFR-3 [vascular endothelial growth factor receptor-3], and Prox-1), with the pan-endothelial markers CD31 and von Willebrand factor (vWF), and with the blood-specific marker CD34. Several blood vessels were identified in human pulps and skin. Lymphatic vessels were found in all human skin samples but in none of the pulps examined. Western blotting performed on human dermis and on pulps treated with collagenase (to remove odontoblasts) confirmed these results. Transmission electron microscopy indicated that vessels which, by light microscopy, appeared to be initial lymphatic vessels had no anchoring filaments or discontinuous basement membrane, both of which are typical ultrastructural characteristics of lymphatic vessels. These results suggest that under normal conditions human dental pulp does not contain true lymphatic vessels. The various theories about dental pulp interstitial fluid circulation should be revised accordingly.

VEGF-A and VEGFR-3 correlate with nodal status in operable non-small cell lung cancer: inverse correlation between expression in tumor and stromal cells.

BACKGROUND: Lymph node metastasis is an essential determinant for stage and clinical management of non-small cell lung cancer (NSCLC). The vascular endothelial growth factors (VEGFs) and receptors (VEGFRs) are fundamental molecules in angiogenesis and lymphangiogenesis. We aimed to explore the correlations between nodal metastasis and the expression of VEGFs and VEGFRs in tumor cells and in tumor-related stroma. PATIENTS AND METHODS: Tumor tissue samples from 335 resected patients with stage I-IIIA NSCLC were obtained and tissue microarrays were constructed from duplicate cores of tumor cells and surrounding stromal tissue from each resected specimen. Immunohistochemistry was used to evaluate the expression of VEGF-A, VEGF-C, and VEGF-D and VEGFR-1, VEGFR-2 and VEGFR-3. RESULTS: There were 232 N0 and 103 N+ patients (76 N1, 27 N2). In multivariate analyses, low stromal VEGF-A expression (P=0.018) is associated with N+ status. In tumor cells, strong correlations exist between high VEGF-A expression (P=0.032) and N+ status, and high VEGFR-3 expression (P<0.001) and N2-status. CONCLUSION: The converse impact by stromal VEGF-A versus tumor cell VEGF-A expression on nodal metastasis may allude the importance of the tumor-stroma interaction when trying to understand lymphatic metastasis in NSCLC.

Vascular endothelial growth factor D is dispensable for development of the lymphatic system.

Vascular endothelial growth factor receptor 3 (Vegfr-3) is a tyrosine kinase that is expressed on the lymphatic endothelium and that signals for the growth of the lymphatic vessels (lymphangiogenesis). Vegf-d, a secreted glycoprotein, is one of two known activating ligands for Vegfr-3, the other being Vegf-c. Vegf-d stimulates lymphangiogenesis in tissues and tumors; however, its role in embryonic development was previously unknown. Here we report the generation and analysis of mutant mice deficient for Vegf-d. Vegf-d-deficient mice were healthy and fertile, had normal body mass, and displayed no pathologic changes consistent with a defect in lymphatic function. The lungs, sites of strong Vegf-d gene expression during embryogenesis in wild-type mice, were normal in Vegf-d-deficient mice with respect to tissue mass and morphology, except that the abundance of the lymphatics adjacent to bronchioles was slightly reduced. Dye uptake experiments indicated that large lymphatics under the skin were present in normal locations and were functional. Smaller dermal lymphatics were similar in number, location, and function to those in wild-type controls. The lack of a profound lymphatic phenotype in Vegf-d-deficient mice suggests that Vegf-d does not play a major role in lymphatic development or that Vegf-c or another, as-yet-unknown activating Vegfr-3 ligand can compensate for Vegf-d during development.