Expression of Vascular Endothelial Growth Factor C and Its Correlation with Lymph Node Metastasis in Colorectal Carcinoma

Summary: To study the expression of vascular endothelial growth factor C (VEGF-C) in colorectal carcinoma and its relationship with lymph node metastasis, the expression of VEGF-C protein in colorectal carcinoma tissues obtained from 94 patients who underwent radical resection was immunohistochemically detected. Meanwhile, the expression of VEGF-C mRNA in 4 colorectal carcinoma cell lines was examined by reverse transcription polymerase chain reaction (RT-PCR).VEGF-C protein was found to be expressed in 53.2 % of patients. The expression was more frequently detected in tumors with lymph node metastasis than in those without metastasis (P<0.01), and there was significant correlation between its expression and lymphatic invasion, TNM stage (P<0.01). However, no significant correlation was found between its expression and the age, gender, tumor location, depth of invasion and vascular invasion. 2 of the 4 colorectal carcinoma cell lines, including LoVo and LoVo-5FU, expressed VEGF-C mRNA. The expression of VEGF-C is closely related to lymph node metastasis, and it might take part in the tumor lymphangiogenesis.

Effect of Antisense Oligodeoxynucleotide of Vascular Endothelial Growth Factor C on Lymphangiogenesis and Angiogenesis of Pancreatic Cancer

In order to investigate the effect of antisense oligonucleotide （ASODN） of vascular endothelial growth factor C （VEGF-C） on lymphangiogenesis and angiogenesis of pancreatic cancer, antisense and scamble-sense oligonucleotide of VEGF-C were constructed, and the model of nude mice with orthotopically xenografied human pancreatic cancer cells （Panc-1） was established. Thirty nude mice were randomly divided into 3 groups： PBS control group （group A）, scramble-sense control group （group B） and antisense group （group C）. All nude mice were treated once every 2 days as 3 times per week, for 3 weeks （oligonucleotide 10 mg/kg every time）. After treatments were completed, ELISA method was used to examine the concentration of VEGF-C in plasma and immunohistochemical method to examine microvessel density （MVD）, lymphtic vessel density （LVD） of pancreatic cancer. The results showed that the expression of VEGF-C was inhibited significantly in group C. The concentrations were 237.5±41.5, 221.5±52.3 and 108.6±14.9 pg/mL in groups A, B and C respectively （P〈0.01）. LVD in groups A, B and C was 13.8±2.1, 12.4±1.9 and 4.2±1.6 respectively （P〈0.01）. MVD in groups A, B and C was 27.5±8.7, 25.9±4.2 and 19.4±5.6 respectively with no significant difference among the groups （P〉0.05）. It was suggested that VEGF-C ASODN decreased the expression levels of VEGF-C in nude mice with orthotopically xenografted human pancreatic cancer, and it could inhibit lymphangiogenesis, but had no significant effect on angiogenesis.

Relationship between the Expression of Vascular Endothelial Growth Factor C and Lymphangiogenesis in Human Pancreatic Cancer

Objective： To investigate the role the vascular endothelial growth factor C （VEGF-C） expression in lymphangiogenesis and lymph node metastasis in pancreatic cancer. Methods： The expression of VEGF-C in pancreatic cancer tissue of 42 cases and normal pancreatic tissue of 10 cases was detected by using immunohistochemical S-P method. The microlymphatic vessel density （MLVD） of both tissues was measured by enzyme-histochemical method at the same time. Results： The VEGF-C positive expression rate was significantly higher in pancreatic cancer tissue （64.3%） than in normal pancreatic tissue （20%, P〈0.05）. The MLVD was significantly higher in pancreatic cancer tissue （21.93±6.39） than that in normal pancreatic tissue （4.40±2.07, P〈0.001）. The VEGF-C positive expression rate, MLVD and lymph node metastasis had a positive correlation. Conclusion： The VEGF-C may play an important role in the lymphangiogenesis and lymph node metastasis of pancreatic cancer.

Correlation between the expression of vascular endothelial growth factor c and C-erbB-2 in human breast cancer

Objective: We aimed to study the transcription level of VEGF-C in human breast cancer tissue, and explore the correlations with the expression of C-erbB-2. Methods: The expression of VEGF-C mRNA in 51 cases of human breast cancer was assessed by hybridization in situ. The expressions of C-erbB-2 was assessed by immunohistochemistry. Results: The positive rate of VEGF-C mRNA was 54.9% in 51 cases of breast cancer. The transcription level had correlation with tumor size and status of lymph nodes(P < 0.05). The expression of VEGF-C mRNA had a positive correlation with the expression of C-erbB-2(P < 0.05). Conclusion: The up-expression of VEGF-C has a significant correlation with the malignancy level and clinical stage of breast cancer. The combined detection of VEGF-C, C-erbB-2 may help to estimate the prognosis of patients with breast cancer and study on thetherapeutic implications.

Expression of Vascular Endothelial Growth Factor C and Its Clinical Significance in Human Esophageal Squamous Cell Carcinoma

OBJECTIVE To examine the expression of vascular endothelial growth factor C （VEGF-C） in human esophageal squamous cell carcinoma （ESCC）, and to clarify its role in lymphatic metastasis in ESCC patients.METHODS Esophageal carcinoma EC9706 cells and samples from 49 patients with primary ESCC were investigated by using S-P immunohistochemistry （IHC）, the semi-quantitative reverse transcriptase-polymerase chain reaction （RT-PCR） and in situ hybridization （ISH） methods for VEGF-C expression. RESULTS VEGF-C positive expression was found in EC9706 cells through IHC, ISH and RT-PCR. Positive IHC for VEGF-C was observed in 36 of 49 cases of ESCC. There was a significant difference between the expression of VEGF-C in a lymph-node-positive group compared to a node-negative group （χ^2=4.7, P〈0.05）. Positive ISH for VEGF-C mRNA was observed in 23 of 49 cases of ESCC. There was a significant difference between the expression of VEGF-C in the lymph-node-positive group and node-negative group （χ^2=31.3, P〈0.01）. The expression of VEGF-C was significantly higher in the lymph-node-positive group compared to the node-negative group. Of 49 ESCC tissues, RT-PCR for VEGF-C mRNA was observed positively in 29 cases. There was a significant difference between the expression of VEGF-C in the lymph-node-positive group and node-negative group （χ^2=23.3, P〈0.01）. The expression of VEGF-C was significantly higher in the lymphnode-positive group compared to the node-negative group. Expressions of VEGF-C were not significantly associated with age, gender, and pathological grade. There was a relationship between VEGF-C mRNA expressions by RT-PCR and ISH （χ^2=18.5, P〈0.01） in ESCC cases, but with no significant difference between the two methods. CONCLUSION VEGF-C expression may induce lymphangiogenesis in human ESCC. There was a close correlation between VEGF-C expression and lymph node metastasis. VEGF-C can serve as a useful prognostic factor for ESCC patients.

Low molecular weight heparin suppresses lymphatic endothelial cell proliferation induced by vascular endothelial growth factor C in vitro

Background Pancreatic cancer is one of the most aggressive human malignancies. Lymphangiogenesis plays an important role in lymph node metastasis of many solid tumors. It is well known that low molecular weight heparins （LMWHs） can inhibit cell growth, cell invasion and angiogenesis, which are key processes in tumor progression. Methods We measured the expression of vascular endothelial growth factor C （VEGF-C） in pancreatic cancer cells （PANC-1） using reverse transcription-polymerase chain reaction （RT-PCR） and Western blotting. We used an in vitro assay to evaluate the anti-lymphangiogenic effect of an LMWH, Fragmin, on human lymphatic endothelial cell （HLEC） proliferation. Results Fragmin at a low concentration can effectively inhibits HLEC proliferation induced by VEGF-C. VEGF-C secreted by PANC-1 cells stimulated HLEC proliferation. Low concentration LMWH suppressed HLEC proliferation induced by VEGF-C but did not affect proliferation or VEGF-C expression of PANC-1 cells, whereas high concentrations of LMWH inhibited PANC-1 cell proliferation. Conclusions These results suggest that VEGF-C released by cancer cells plays an important role in promoting HLEC proliferation. The LMWH Fragmin has anti-lymphangiogenic effects and may inhibit lymphatic metastasis in pancreatic cancer.

Serum Vascular Endothelial Growth Factor-C and Vascular Endothelial Growth Factor Level in Patients with Colorectal Carcinoma and Clinical Significance

Circulating vascular endothelial growth factor-C （VEGF-C） and vascular endothelial growth factor （VEGF） levels in patients with colorectal carcinoma were determined in order to assess their clinical significance as a diagnostic tool for monitoring lymph node metastasis. In 66 patients with colorectal carcinoma and 30 healthy controls, circulating VEGF-C and VEGF levels were assessed by using enzyme-linked immunosorbent assay （ELISA）. Serum VEGF-C and VEGF levels were higher in patients with colorectal carcinoma than in healthy controls. Patients with lymph node metastasis had higher serum VEGF-C and VEGF levels than those without lymph node metastasis. The levels of VEGF-C and VEGF were higher in the invasion group than in the non-invasion group. Serum VEGF-C levels reached a sensitivity of 81% and a specificity of 76 % with a cutoff value of 1438.0 pg/mL, whereas VEGF levels reached 72 % sensitivity and 74 % specificity at 240.2 pg/ mL. If 66 patients were divided into 4 groups according to the combined determination of VEGF-C and VEGF levels, the positive predictive value was 85.3 %, the negative predictive value was 94.6 %, and accuracy was 93.7 %. It was suggested that circulating VEGF-C levels might provide additional information for distinguishing the absence from presence of lymph node metastasis in patients with colorectal carcinoma. The combined determination of VEGF-C and VEGF levels could be used as an important index for preoperatively clinical stage of colorectal carcinoma.

Increased expressions of vascular endothelial growth factor(VEGF),VEGF-C and VEGF receptor-3 in prostate cancer tissue are associated with tumor progression

Aim： To investigate the differences in microvessel densities （MVD） and the expressions of vascular endothelial growth factor （VEGF）, VEGF-C and VEGF receptor-3 （VEGFR-3） between prostate cancer （PCa） tissues and adjacent benign tissues, and to explore the correlations among MVD, Jewett-Whitmore staging, Gleason scores and expressions of VEGF, VEGF-C and VEGFR-3 in the progression of PCa. Methods： An immunohistochemical approach was adopted to detect the expressions of CD34, VEGF, VEGF-C and VEGFR-3 in both cancer areas and peripheral benign areas of 71 primary prostatic adenocarcinoma specimens. A statistic analysis was then performed according to the experimental and clinic data. Results： Significantly upregulated expressions of VEGF, VEGF-C and VEGFR-3 were all found in malignant epithelium/cancer cells compared with adjacent benign epithelium （P 〈 0.01）. Patients in stage D had a significantly higher score than patients in stage A, B or C when comparing the expression of VEGF-C or VEGFR-3 in the tumor area （P 〈 0.01）. In addition, significant correlations were observed between Jewett-Whitmore staging and VEGF-C （rs = 0.738, P 〈 0.01）, clinical staging and VEGFR-3 （rs = 0.410, P 〈 0.01）, VEGF-C and Gleason scores （rs = 0.401, P 〈 0.01）, VEGFR-3 and Gleason scores （rs = 0.581, P 〈 0.001） and MVD and VEGF （rs = 0.492, P 〈 0.001）. Conclusion： Increased expressions of VEGF and VEGF-C were closely associ- ated with progression of PCa. The main contribution of increased VEGF expression for PCa progression was to upregulate MVD, which maintained the growth advantage of tumor tissue. However, the chief role of increased expressions of VEGF-C and VEGFR-3 was to enhance lymphangiogenesis and provide a main pathway for cancer cells to disseminate. （Asian J Androl 2006 Mar; 8： 169-175）

Epithelial membrane protein 1 negatively regulates cell growth and metastasis in colorectal carcinoma

AIM: To determine the expression and function of epithelial membrane protein 1 (EMP1) in colorectal carcinoma.

The lymphatic system:a therapeutic target for central nervous system disorders

The lymphatic vasculature forms an organized network that covers the whole body and is involved in fluid homeostasis,metabolite clearance,and immune surveillance.The recent identification of functional lymphatic vessels in the meninges of the brain and the spinal cord has provided novel insights into neurophysiology.They emerge as major pathways for fluid exchange.The abundance of immune cells in lymphatic vessels and meninges also suggests that lymphatic vessels are actively involved in neuroimmunity.The lymphatic system,through its role in the clearance of neurotoxic proteins,autoimmune cell infiltration,and the transmission of pro-inflammatory signals,participates in the pathogenesis of a variety of neurological disorders,including neurodegenerative and neuroinflammatory diseases and traumatic injury.Vascular endothelial growth factor C is the master regulator of lymphangiogenesis,a process that is critical for the maintenance of central nervous system homeostasis.In this review,we summarize current knowledge and recent advances relating to the anatomical features and immunological functions of the lymphatic system of the central nervous system and highlight its potential as a therapeutic target for neurological disorders and central nervous system repair.

Expression of vascular endothelial growth factor （VEGF） and VEGF-C in serum and tissue of Wilms tumor

Background Angiogenesis and lymphogenesis which were promoted by vascular endothelial growth factor （VEGF） and VEGF-C are important in the growth and metastasis of solid tumors. The high level of VEGF and VEGF-C were distributed in numerous types of cancers, but their distribution and expression in Wilms tumor, the most common pediatric tumor of the kidney, was unclear. Methods To learn about the distribution, mass spectroscopy and immunohistochemistry were used to measure the level of VEGF and VEGF-C in serum and tissue of Wilms tumor. Results The expression level of VEGF in serum of Wilms tumor was the same as in pre-surgery and control, so it was the same case of VEGF-C. Both of these factors were chiefly located in Wilms tumor tissue, but not in borderline and normal. In addition, the higher clinical staging and histopathologic grading were important elements in high expression of VEGF and VEGF-C. Gender, age and the size of tumor have not certainly been implicated in expression level of VEGF and VEGF-C. Conclusions The lymph node metastasis and growth of tumors resulted from angiogenesis and lymphogenesis which were promoted by VEGF and VEGF-C in Wilms tumor. The autocrine and paracrine process of VEGF and VEGF-C were the principal contributor to specific tissues of Wilms tumor but not to the entire body.

Effect of HIF-1αon VEGF-C Induced Lymphangiogenesis and Lymph Nodes Metastases of Pancreatic Cancer

The effect of hypoxia inducible factor-1 α （HIF-1 α） on vascular endothelial growth factor C （VEGF-C） and the correlation between HIF- 1 α and lymphangiogenesis and lymph nodes metastases （LNM） in pancreatic cancer were investigated. Immunohistochemical SP method was used to detect the protein expression of HIF-1 α and VEGF-C, and Lymphatic vessel density （LVD） was determined by stain of VEGFR-3, collagen type IV in 75 pancreatic head cancers from regional pancreatectomy （RP） during Dec. 2001 to Dec. 2003. The relationship between HIF-1α and VEGF-C, lymphangiogenesis, LNM was analyzed statistically. The results showed that the positive expression rate of HIF-1α and VEGF-C in pancreatic cancer tissues was 48.00 % （36/75） and 65.33 % （49/75） respectively. In positive group of HIF-1α, the positive rate of VEGF-C and LVD, and LVD rate was 80.56 % （29/36）, 13.22±3.76 and 88.89 % （32/36） respectively, and in negative group of HIF-10t, positive rate of VEGF-C and LVD was 51.28 % （20/39）, 5.98±2.17 and 66.67 % （26/39） respectively （P〈0.01 or P〈0.05）. It was suggested that HIF-1α could promote the expression of VEGF-C, lymphangiogenesis and LNM in pancreatic cancer.

Expression of VEGF-C in Rat Cornea after Alkali Injury

The expression of VEGF-C and molecular mechanisms of lymphangiogenesis in rat cornea after alkali injury was studied. The rat alkali injured corneal models were made. Under electron microscopy, the lymphatic vessels in the rat injured corneas were examined. The expression of VEGF-C proteins was detected by using immunohistochemical assay at day 1, 3, 5, 7, 9 after injury. The expression levels of VEGF-C mRNA were quantified with reverse transcription polymerase chain reaction (RT-PCR). The results showed that the lymphatic vessels were found in the injured rat corneas 14 days after the injury. The VEGF-C protein was detectable 3 days after injury, reached the peak 5 days after injury, and gradually decreased. In the control group, no VEGF-C proteins were detected. The VEGF-C mRNA was minimally detected in the normal rat corneas, but it was highly expressed 5 days after the injury. The difference was statistically significant. It was concluded that VEGF-C might be one of the most important relevant factors in corneal lymphangiogenesis after alkali injury.

Effects of carbon dioxide pneumoperitoneum on metastasis of ovarian carcinoma cell line SKOV_3

The long suspicion of the potential harm of carbon dioxide （CO2） pneumoperitoneum exists in laparoscopic cancer surgery. For better understanding of this problem, we targeted this study at the effects of CO2 pneumoperitoneum on the invasive ability of ovarian carcinoma cell line and the possible mechanism within it. To study the effects of CO2 pneumoperitoneum on carcinoma cell, SKOV3 cells were divided into 2 groups, respectively exposed to pneumoperitoneal CO2-insuffiation and normal conditions. To study the possible mechanism, SKOV3 cells were divided into 3 groups, one was exposed to CO2 pneumoperitoneum, one to N2 and the other to normal conditions served as control. The in vitro adhesive and invasive ability of the cells was analyzed through 3-（4,5-dimethylthiazol-2-yl）-2,5-diphenyl tetrazolium bromide （MTT） assay and Boyden filters metastasis model; the expressions of vascular endothelial growth factor C （VEGF-C） and matrix metalloproteinase 9 （MMP9） were determined by reverse transcription polymerase chain reaction（RT-PCR）, and Western blot. We found that the adhesive ratio of SKOV3 cells exposed to CO2 was significantly higher than that of the control group; cells exposed to CO2 invaded the matrigel with a greater number （P〈0.01）; the expression of VEGF-C exposed to both CO2 and N2 was significantly increased compared with control group （P〈0.05）; the MMP9 expression level of CO2 group was higher than that of N2 group, P〈0.05. We concluded that carbon dioxide pneumoperitoneum may improve the adhesive and invasive ability of ovarian carcinoma cell line in vitro and CO2 can also be an independent factor to stimulate the expression of MMP9.

Meningeal lymphatic vasculature,a general target for glioblastoma therapy?

Glioblastoma(GBM)causes nearly universal mortality as a result of the failure of conventional therapies including surgical resection,targeted radiation therapy,and chemotherapy.An increasingly important treatment option is combining immunotherapy with other therapies in both preclinical and clinical studies.The central nervous system(CNS)has been historically considered an immune privileged area,but increasing evidence,including the recent rediscovery of meningeal lymphatic vessels(MLVs),has overturned this notion.MLVs are populated by multiple immune cells and connect the CNS to the periphery by draining cerebrospinal fluid with soluble CNS antigens and immune cells into cervical lymph nodes.In the past few years,more and more studies have indicated that MLVs are involved in the regulation of inflammation and the immune response in the pathogenesis of various CNS diseases including GBM.Here,we explore the critical interlinkages between MLVs and GBM therapies including chemotherapy,radiotherapy and immunotherapy,and propose the meningeal lymphatic vasculature as a general target for GBM therapy.

Knockdown of Decoy Receptor 3 Impairs Growth and Invasiveness of Hepatocellular Carcinoma Cell Line of HepG2

Background：Decoy receptor 3 （DcR3） binds to Fas ligand （FasL） and inhibits FasL-induced apoptosis.The receptor is overexpressed in hepatocellular carcinoma （HCC）,and it is associated with the growth and metastatic spread of tumors.DcR3 holds promises as a new target for the treatment of HCC,but little is known regarding the molecular mechanisms underlying the oncogenic properties of DcR3.The present work,therefore,examined the role of DcR3 in regulating the growth and invasive property of liver cancer cell HepG2.Methods：HepG2 cells were stably transfected with lentivirus-based short hairpin RNA vector targeting DcR3.After the knockdown of DcR3 was confirmed,cell proliferation,clone formation,ability of migrating across transwell membrane,and wound healing were assessed in vitro.Matrix metalloproteinase-9 （MMP 9） and vascular epithelial growth factor （VEGF）-C and D expressions of the DcR3 knockdown were also studied.Comparisons between multiple groups were done using one-way analysis of variance （ANOVA）,while pairwise comparisons were performed using Student＇s t test.P 〈 0.05 was regarded statistically significant.Results：DcR3 was overexpressed in HepG2 compared to other HCC cell lines and normal hepatocyte Lo-2.Stable knockdown of DcR3 slowed down the growth of HepG2 （P 〈 0.05） and reduced the number of clones formed by 50% compared to those without DcR3 knockdown （P 〈 0.05）.The knockdown also reduced the migration of HepG2 across transwell matrix membrane by five folds compared to the control （P 〈 0.05） and suppressed the closure of scratch wound （P 〈 0.05）.In addition,the messenger RNA levels of MMP 9,VEGF-C,and VEGF-D were significantly suppressed by DcR3 knockdown by 90% when compared with the mock control （P 〈 0.05）.Conclusions：Loss of DcR3 impaired the growth and invasive property of HCC cell line of HepG2.Targeting DcR3 may be a potential therapeutic approach for the treatment of HCC.

Efficacy and effect on serum VEGF-C of mild moxibustion plus functional exercise for upper-limb lymphedema after breast cancer surgery

Objective To observe the efficacy of mild moxibustion combined with functional exercise in the treatment of upper-limb lymphedema after breast cancer surgery and its effect on serum vascular endothelial growth factor C(VEGF-C).Methods Seventy-eight patients were divided into a control group and an observation group by the random number table method,with 39 cases in each group.The control group received functional exercise and the observation group received mild moxibustion plus functional exercise.The differences in circumference between the two upper limbs,the lymphatic flow of the affected upper limb,the disability of arm,shoulder and hand(DASH)score,the functional assessment of cancer therapy-breast(FACT-B)score and serum VEGF-C level between the two groups were compared before and after treatment.Efficacy was evaluated after treatment.Results The total effective rate was significantly higher in the observation group than in the control group(P<0.05).The difference in circumference between the two upper limbs of the two groups decreased significantly after treatment(both P<0.05),but it was significantly lower in the observation group than in the control group(P<0.05).The lymphatic flow of the affected upper limb of the two groups increased significantly after treatment(both P<0.05),but it was significantly greater in the observation group than in the control group(P<0.05).The DASH scores of the two groups decreased significantly after treatment(both P<0.05),but it was significantly lower in the observation group than in the control group(P<0.05).The FACT-B scores of the two groups increased significantly after treatment(both P<0.05),but it was significantly higher in the observation group than in the control group(P<0.05).After treatment,the serum VEGF-C level increased significantly in the observation group(P<0.05),whereas the control group did not show significant change(P>0.05).The post-treatment serum VEGF-C level was significantly higher in the observation group than in the control group(P<0.05).Conclusion The efficacy of mild moxibustion combined with functional exercise for upper-limb lymphedema after breast cancer surgery is certain,which can reduce the difference in circumference between the two upper limbs,increase the lymphatic flow of the affected upper limb,improve the limb function and the quality of life,and regulate the serum VEGF-C level.

Coexpression of TLR9 and VEGF-C is associated with lymphatic metastasis in prostate cancer

Prostate cancer(PCa)is one of the most frequent cancers in men,and its biomolecular targets have been extensively studied.This study aimed to analyze the expression of toll-like receptor 9(TLR9)and vascular endothelial growth factor C(VEGF-C)and the clinical value of the coexpression of TLR9 and VEGF-C in PCa.We retrospectively evaluated 55 patients with clinically localized,intermediate-risk,or high-risk PCa who underwent laparoscopic radical prostatectomy(LRP)and extended pelvic lymph node dissection(ePLND)without neoadjuvant hormonal therapy at a single institution from June 2013 to December 2016.In all 55 patients,the median number of lymph nodes(LNs)resected was 23(range:18-31),and a total of 1269 LNs were removed,of which 78 LNs were positive.Seventeen patients had positive LNs,with a positive rate of 30.9%.In addition,the immunohistochemical results in the above patients revealed that high TLR9 expression was correlated with higher Gleason score(GS)(P=0.049),increased LN metastasis(P=0.004),and more perineural invasion(PNI)(P=0.033).Moreover,VEGF-C expression was associated with GS(P=0.040),pathological stage(pT stage)(P=0.022),LN metastasis(P=0.003),and PNI(P=0.001).Furthermore,a significant positive correlation between TLR9 and VEGF-C was found(P<0.001),and the TLR9/VEGF-C phenotype was associated with LN metastasis(P=0.047).Collectively,we propose that TLR9 stimulation may promote LN metastasis in PCa cells through the upregulation of VEGF-C expression,thereby affecting the prognosis of PCa patients.Therefore,these markers may serve as valuable targets for the treatment of PCa.