Expression of Vascular Endothelial Growth Factor-C and Vascular Endothelial Growth Factor Receptor-3 in Ovarian Epithelial Tumors

Objective： 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. Methods： In situ hybridization and immunohistochemical staining for VEGF-C were performed in 30 epithelial ovarian carcinomas, 9 borderline tumors and 26 benign tumors. 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. Results： VEGF-C mRNA and protein expression were detected in cytoplasm of carcinoma cells. VEGF-C mRNA and protein expression in ovarian epithelial carcinomas were significantly higher than those in borderline tumors and benign tumors （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 lymph node metastasis than those of clinical stage Ⅰ-Ⅱ and without lymph node metastasis respectively （P〈0.05 or P〈0.01）. VEGFR-3 positive vessels and MVD were significantly higher in VEGF-C protein positive tumors than negative tumors （P〈0.05）. VEGFR-3 positive vessels was significantly correlated with MVD（P〈0.01）. Conclusion： VEGF-C might play a role in lymphatic metastasis via lymphangiogenesis and angiogenesis in epithelial ovarian tumors, and VBEGF-C could be used as a biologic marker of metastasis in ovarian epithelial tumors.

Expression of vascular endothelial growth factor-C and angiogenesis in esophageal squamous cell carcinoma

AIM： To investigate the expression of vascular endothelial growth factor-c （VEGF-C） mRNA and microvessel density （MVD） in human esophageal squamous cell carcinoma （ESCC） and its relationship with clinical significance. METHODS： Specimens obtained from 43 patients undergoing surgical resection for ESCC were used in this study. The expression of VEGF-C mRNA was examined by in situ hybridization. Tumor MVD was determined immunohistochemically with anti-CD31 antibody and estimated by image analysis. Ten sections of adjacent normal mucosa were also examined. RESULTS： VEGF-C mRNA expression was detected in cytoplasm of carcinoma cells. Of the 43 ESCC patients studied, 18 cases （41.9%） were positive for VEGF-C mRNA. No VEGF-C mRNA expression was observed in normal esophageal mucosa. VEGF-C mRNA expression correlated significantly with lymph node metastasis, TNM stage and depth of invasion （P 〈 0.05）. Furthermore, histological grade （differentiation） tended to correlate with VEGF-C mRNA expression, but was not statistically significant （P 〉 0.05）. In tumor lesions, the MVD was significantly greater than that in normal esophageal mucosa. MVD correlated significantly with lymph node metastasis, TNM stage and depth of invasion （P 〈 0.05）, but not with histological grade （differentiation） （P 〉 0.05）. Lesions with VEGF-C mRNA expression had a significantly higher MVD than that of those without VEGF-C mRNA expression （P 〈 0.05）. CONCLUSION： VEGF-C plays a role in lymphatic metastasis via lymphangiogenesis and angiogenesis in ESCC. VEGF-C is one of the important predictors of the biological behavior in ESCC.

Expression of vascular endothelial growth factors A and C in human pancreatic cancer

AIM： To study the expression of vascular endothelial growth factor A （VEGF-A） and VEGF-C and to determine whether the presence of VEGF-A and VEGF-C was associated with the clinicopathologic characteristics of pancreatic cancer. METHODS： VEGF-A and VEGF-C mRNA transcripts were examined by Northern blot in 6 human pancreatic cancer cell lines and 8 normal pancreatic tissues and 8 pancreatic carcinoma specimens. The expression of VEGF-A and VEGF-C proteins was examined by Western blot in the tested cell lines and by immunohistochemical stain in 50 pancreatic carcinoma samples. RESULTS： VEGF-A and VEGF-C mRNA transcripts were present in all the 6 human pancreatic cancer cell lines. Immunoblotting revealed the presence of VEGF-A and VEGF-C proteins in all the cell lines. Northern blot analysis of total RNA revealed 3.0-fold and 3.6-fold increase in VEGF-A and VEGF-C mRNA transcript in the cancer samples, respectively. Immunohistochemical analysis confirmed the expression of VEGF-A and VEGF-C in cancer cells within the tumor mass. Immunohistochemical analysis of 50 pancreatic cancer tissue samples revealed the presence of VEGF-A and VEGF-C immunoreactivity in 50% and 80% of the cancer tissue samples, respectively. The presence of VEGF-A in these cells was associated with larger tumor size and enhanced local spread （x^2= 6.690, P= 0.035〈0.05） but was not associated with decreased patient survival. However, the presence of VEGF-C in the cancer cells was associated with increased lymph node metastasis （x^2= 5.710, P= 0.017〈0.05）,but was not associated with decreased patient survival. There was no correlation between the expression of VEGF-A and VEGF-C in the same cancer cells. CONCLUSION： VEGF-A and VEGF-C are commonly overexpressed in human pancreatic cancer and may contribute to tumor growth and lymph node metastasis, There is no relationship between the expression of VEGF-A and VEGF-C in pancreatic cancer.

Interleukin-1α, 6 regulate the secretion of vascular endothelial growth factor A, C in pancreatic cancer

Vascular endothelial growth factor (VEGF, namely VEGF-A) is an angiogenic polypeptide and VEGF-C is a lymphangiogenic polypeptide that has been implicated in cancer growth, invasion and metastasis. Several cytokines and growth factors play an important part in cancer progression. These cytokines and growth factors are the principal mediators of cancer cells-stromal cell interaction , which is critical for invasion of cancer cells to the surrounding tissues and metastatic dissemination to distant organs. In this study, we studied VEGF-A, C expression in cultured human pancreatic cancer cell lines and whether the presence of VEGF-A, C in the cell lines is regulated by cytokines interleukin-lct (EL-1α), and interleukin-6 (IL-6). METHODS: We used Northern blot and Western blot methods to analyze expression of the gene and protein of VEGF-A, C in all 6 tested cell lines (ASPC-1, CAPAN-1, MIA-PaCa-2, PANC-1, COLO-357 and T3M4) respectively. To analyze what is the regulator for this VEGF-A, C expression in pancreatic cancer,we used the reverse transcription -polymerase chain reaction (RT-PCR) method to analyze VEGF-A, C expression in cultured human pancreatic cancer cell lines (CAPAN-1 and COLO-357) under the stimulation with IL-1α (10μg/L) or IL-6 (100 μg/L). RESULTS:Northern blot analysis revealed the presence of the 4.1-kb VEGF-A mRNA transcript and 2.4-kb VEGF-C mRNA transcript in all 6 tested cell lines. Immunoblotting with highly specific anti-VEGF-A, anti-VEGF-C antibody revealed the presence of a molecular weight of 43-kDa VEGF-A protein and 55-kDa VEGF-C protein in all the cell lines. RT-PCR analysis revealed the levels of the VEGF-A and VEGF-C gene were 1-2 fold and a 1-fold increase in the COLO-357 cell line by stimulation with IL-la, however, no effect was found in the CAPAN-1 cell line. The levels of the VEGF-A and VEGF-C gene were 2-5 fold and a 1-fold increase in the CAPAN-1 cell line by stimulation with IL-6, but, no effect was found in the COLO-357 cell line. CONCLUSION:These findings suggested that the expression of VEGF-A, C and their regulation by IL-1α, IL-6 in pancreatic cancer contributes to the lymphatic and distant metastasis and the disease progression.

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.

Gecko crude peptides inhibit migration and lymphangiogenesis by down regulating the expression of VEGF-C in human hepatocellular carcinoma cells and human lymphatic endothelial cells

OBJECTIVE To explore the role of gecko crude peptides(GCPs)in the proliferation,apoptosis,migration and lymphangiogenesis of human hepatocellular carcinoma cells(Hep G2)and human lymphaticendothelial cells(HLECs)in vitro.METHODS The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT)assay was used to evaluate the anti-proliferative effect of GCPs and si RNA-VEGF-C on Hep G2 cells,Hoechst 33258 staining and flow cytometry were performed to analyze cycle and apoptosis.The migration and invasion ability of cells were assayed by transwell chamber experiment and wound-healing assay.The protein and m RNA expressions of vascular endothelial growth factor-C(VEGF-C)and CXC chemokine receptor-4(CXCR4)were detected by q-PCR,immunofluorescence,Western blot.The protein expressions of the extracellular signal regulated kinase(ERKI/2),c-Jun N-terminal kinase(JNK),p38-mitogen activated protein kinases(p38 MAPK),serine/threonine kinase(Akt)and phosphatidylinositol-3-kinase(PI3K)were detected by western blot.The anti-lymphangiogenesis effect of GCPs on the HLECs was analyzed using an in vitro tube-formation assay.The protein and m RNA expressions of vascular endothelial growth factor receptor-3(VEGFR-3)and stromal cell-derived factor-1(SDF-1)were detected by q-PCR,Western blot.RESULTS GCPs and si RNA-VEGF-C inhibited Hep G2 proliferation,invasion and migration,and the most obvious inhibitory effect was both synergistic effects.Thus,GCPs suppressed HLECs proliferation,migration and tubelike structure formationin a dose-dependent manner,and had inhibitory effect of tumor-induced lymphangiogenesis in vitro.Additionally,we found that GCPs and si RNA-VEGF-C decreased the expressions of MMP-2,MMP-9,VEGF-C,CXCR4,phospho-ERK1/2,phospho-P38,phospho-JNK and PI3K in Hep G2 cells.Moreover,GCPs had a dose-dependent depressive effecton the expressions of VEGFR-3,SDF-1 in HLECs.CONCLUSION The low expression of VEGF-C mediated by si RNA-VEGF-C and GCPs inhibit tumor proliferation,invasion and migrationby suppressing the MAPK signaling pathway through reduced levels of VEGF-C,and GCPs inhibit tumor lymphangiogenesis by suppressing the CXCR4/SDF-1 signaling pathway through suppressed VEGF-C/VEGFR-3.

Vector-based RNA interference against vascular endothelial growth factor-C inhibits tumor lymphangiogenesis and growth of colorectal cancer in vivo in mice

Background RNA interference （RNAi） technology is emerging as a very potent tool to generate a cellular knockdown of a desired gene. The aim of this study was to explore whether RNAi targeting vascular endothelial growth factor-C （VEGF-C） could inhibit colorectal tumor lymphangiogenesis and tumor growth. Methods We used vector-based RNAi to inhibit VEGF-C expression in colon cancer in vitro and in vivo. VEGF-C expression was quantified by real-time polymerase chain reaction and Westen blotting. To establish LoVo cell tumor xenografts in mice, we subcutaneously inoculated 1.0×10^6 LoVo cells in nude mice; after injection, tumors were allowed to grow for 4 weeks until the volume reached （75.80± 55.8）mm^3. The mice were then randomly divided into two groups： （1） the VEGF-C-siRNA group （n= 10） received direct injection of ＂therapeutic＂ plasmid 50 IJg of LoVo-VEGF-C-siRNA into the tumor mass; （2） the control group （n= 10） were injected with LoVo-control in 20 IJI of sterile 0.9% NaCI solution into the tumor mass. Tumor growth, microlymphatics and microvessels were compared for mice administered either systemic VEGF-C-siRNA or control over 4 weeks. Results The mRNA and protein expression of VEGF-C in the colon tumor cell line, LoVo, stably transfected with a VEGF-C-siRNA vector, were significantly downregulated 45.3% and 35.3% respectively. In vivo, four weeks after injection, the tumor volume were significantly smaller in VEGF-C-siRNA group than in LoVo-control group （（314.8 ± 54.8） mm3 vs （553.9 ± 90.1） mm3）; the incidences of lymph node metastasis （30%） in VEGF-C-siRNA were significantly inhibited compared with LoVo-control group （70%）; in VEGF-C-siRNA group, the number of microlymphatics per microscopic field was （5.3 ± 0.7） and the number of microvessels per microscopic field was （24.2 ± 6.5） decreased compared with LoVo-control group （12.5 ± 6.9） and （42.1 ± 7.4） （all P〈0.001）. Conclusion Inhibition of VEGF-C expression using siRNA-mediated gene silencing vectors reduced lymphangiogenesis and tymph node metastasis and enhanced survivat.

Stable transfection of estrogen receptor-alpha suppresses expression of cyclooxygenase-2 and vascular endothelial growth factor-C in MDA-MB-231 breast cancer cells

Background Estrogen receptor （ER）-negative breast cancer cells are more aggressive than ER-positive cells. Elevated levels of cyclooxygenase-2 （COX-2） and vascular endothelial growth factor-C （VEGF-C） expression have been detected in cultured human breast cancer cells and are associated with negative hormone receptor status. In this study, we created ERα stable transfectants in MDA-MB-231 cells to explore the effect of ERα on cell growth and COX-2 and VEGF-C expression.Methods The green fluorescent protein （GFP）-ERα plasmids were stably transfected into ER-negative MDA-MB-231 cells. The proliferation and migration of untransfected MDA-MB-231 cells, ERα-transfected MDA-MB-231 cells and ER-positive MCF-7 cells were determined. The expression of COX-2, and the levels of VEGF-C mRNA and the VEGF-C secretion concentration were assayed in these cell lines.Results The proliferation and migration capacities of ERα-tranfected MDA-MB-231 cells were significantly decreased （P 〈0.05）. The expression of COX-2 was significantly lower in ERa-tranfected MDA-MB-231 cells than in untranfected MDA-MB-231 cells. The mRNA and protein levels of VEGF-C were lower in ERa-tranfected MDA-MB-231 cells than in untransfected MDA-MB-231 cells （P〈0.05）.Conclusions ERα stable transfection inhibits proliferation and migration capacities of MDA-MB-231 cells and decreases expression of COX-2 and VEGF-C. The decreases of proliferation and migration capacities may be related to suppression of COX-2 and VEGF-C expression.

Metastasis-associated protein 1 induces VEGF-C and facilitates lymphangiogenesis in colorectal cancer

AIM:To study the correlation between high metastasisassociated protein 1(MTA1)expression and lymphangiogenesis in colorectal cancer(CRC)and its role in production of vascular endothelial growth factor-C(VEGF-C). METHODS:Impact of high MTA1 and VEGF-C expression levels on disease progression and lymphovasculardensity(LVD,D2-40-immunolabeled)in 81 cases of human CRC was evaluated by immunohistochemistry. VEGF-C mRNA and protein expressions in human LoVo and HCT116 cell lines were detected by real-time polymerase chain reaction and Western blotting,respectively,with a stable expression vector or siRNA. RESULTS:The elevated MTA1 and VEGF-C expression levels were correlated with lymph node metastasis and Dukes stages(P<0.05).Additionally,high MTA1 expression level was correlated with a large tumor size(P< 0.05).A significant correlation was found between MTA1 and VEGF-C protein expressions in tumor cells(r=0.371, P<0.05).Similar to the VEGF-C expression level,high MTA1 expression level was correlated with high LVD in CRC(P<0.05).Furthermore,over-expression of MTA1 significantly enhanced the VEGF-C mRNA and protein expression levels,whereas siRNAs-knocked down MTA1 decreased the VEGF-C expression level. CONCLUSION:MTA1,as a regulator of tumor-associated lymphangiogenesis,promotes lymphangiogenesis in CRC by mediating the VEGF-C expression.

Clinical Significance of VEGF-C and VEGFR-3 Expression in Non-small Cell Lung Cancer

The relationship between vascular endothelial growth factor-C （VEGF-C）/vascular endothelial growth factor receptor 3 （VEGFR-3） expression and clinicopathologic features of the patients with non-small cell lung cancer （NSCLC） was investigated. The expression of VEGF-C and VEGFR-3 was assessed in 65 patients with NSCLC by immunohistochemistry. The significance of VEGF-C and VEGFR-3 expression was analyzed statistically. The results showed that VEGF-C and VEGFR-3 were highly expressed in cytoplasm and membrane in lung cancer tissues with the positive rate being 55.4 % and 52.3 % respectively, while there was no expression in the normal lung tissues. The expression of VEGF-C was significantly increased in adenocacinoma as compared to other types of NSCLC （P〈0.05）. The VEGFR-3 expression was closely related with lymph node metastasis （P〈0.01） and TNM stage （P〈0.05）. There was a positive correlation between the VEGF-C and VEGFR-3 expression in NSCLC patients （r=-0.658, P〈0.01）. It is suggested that VEGFR-3 plays an important role in the lymphatic metastasis of NSCLC. The interaction between VEGF-C and VEGFR-3 may be deeply involved in the mechanism of lung cancer metastasis.

The Expression of VEGF-C and It’s Receptor VEGFR-3 Correlates with Lymph Node Metastasis in Gastric Cancer

Background: Regional lymph node invasion and metastasis may happen early during the progres-sion of gastric cancer. The lymphadenectomy is still the key method to treat lymph nodemetastasis. In the recent years, scientists have found some growth factors and receptors that can promote angiogenesis which also play an important role in adjusting the formation of the new lymph vessel, and lymphangiogenesis may play a key role in the process of lymph node metastasis. Objectives: This study aims to explore the correlation between the expression of vascular endothelial growth factor-C (VEGF-C), vascular endothelial growth factor receptor 3 (VEGFR-3) and lymph node me-tastasis (LNM), and its impact on prognosis of patients with gastric cancer. Methods: The samples were collected from gastric cancer database of Sichuan Provincial People’s Hospital from 2005 to 2007, which were registered and followed up. The samples were divided into two groups according to situation whether there is lymph node metastasis, which is lymph node metastasis N(+) and without lymph node metastasis N(﹣). The expression of VEGF-C, VEGFR-3 and CD34 were measured by immuno histochemistry staining with monoclonal antibody (anti-VEGF-C, anti-VEGFR-3, and anti-CD34). Kaplan-meier, logistic and Cox regression was performed to explore their impact on the prognosis of patients with gastric cancer. Results: In total 186 cases were collected, 96 cases in N(+) group and 90 cases in N(﹣) group. The percentage of VEGF-C expression is 54.83% (102/186) in all groups, 73.9% (71/96) in N(+) group, and 34.44% (31/90) in N(﹣) group (p = 0.001). The percentage of VEGFR-3 expression is 33.33% (62/186) in all groups, 44.78% (43/96) in N(+) group, and 21.11% (19/90) in N(﹣) group (p = 0.001). There are no statistical differences in microvessel density (MVD) between N(﹣) and N(+) group. The average lymphatic vessel density (LVD) was significant different between N(+) and N(﹣) group (26.23 ± 8.2 and 18.46 ± 7.4, t = ﹣2.427, p = 0.016). The five-year overall survival rate of N(+) group is 31% and the N(﹣) group is 66%;there are statistical differences between the two groups (Log rank = 27.15, p = 0.001). The five-year overall survival rates of VEGF-C positive group and VEGF-C negative group are 36% and 59%, with the statistical differences (Log rank = 27.15, p = 0.001). And the five-year overall survival rates of VEGFR-3 positive group and VEGFR-3 negative group are 31% and 43%, also with the statistical differences (Log rank = 5.241, p = 0.041). Conclusions: The expressions of VEGF-C, VEGFR-3 in cell plasma of gastric cancer tissue not only correlate with lymphatic vessel density and lymph node metastasis (LNM), but also are important factors which impact prognosis of gastric cancer patients.

Effect of epidural analgesia and intravenous analgesia on serum CA125, VEGF-C and PGE2 in patients with hysteromyoma

Objective:To investigate the effects of epidural analgesia and intravenous analgesia on serum Cancer 125 (antigen 125, CA125), vascular endothelial growth factor-C (Vascular endothelial growth factor-C, VEGF-C) and prostaglandin E2 (Prostaglandin VEGF-C) in patients with uterine fibroids.Methods:A total of 98 cases of uterine myoma treated in our hospital during September 2017 in September 2015 were selected and divided into 49 cases in the observation group and 49 cases in the control group according to the random table method. The two groups were all anaesthetized by subarachnoid block. The control group was controlled by self controlled intravenous analgesia, and the observation group was treated with epidural anesthesia. The operation time and the amount of intraoperative bleeding were compared between the two groups, and the changes of preoperative and postoperative 48 h VAS score, CA125, VEGF-C and PGE2 levels.Results: There was no statistical difference between the two groups of operation time and intraoperative bleeding. Two groups of postoperative 3 h, postoperative 24 h and 48 h VAS score decreased compared with preoperative;the observation group 3 h, 24 h and 48 h VAS scores were lower than those in the control group. Two groups of postoperative 48 h serum CA125, VEGF-C and PGE2 levels decreased;postoperative 48 h serum CA125, VEGF-C and PGE2 levels after operation in the observation group lower than in the control group.Conclusion: The effect of epidural analgesia on uterine myoma is obvious, and it can reduce the level of CA125, VEGF-C and PGE2, which is of great significance.

VEGF-D expression correlates with colorectal cancer aggressiveness and is downregulated by cetuximab

AIM:To gain mechanistic insights into the role played by epidermal growth factor receptor (EGFR) in the regulation of vascular endothelial growth factors (VEGFs) in colorectal cancer (CRC). METHODS:The impact of high-level expression of the growth factor receptors EGFR and VEGF receptor (VEGFR)3 and the VEGFR3 ligands VEGF-C and VEGF-D on disease progression and prognosis in human CRC was investigated in 108 patients using immunohistochemistry. Furthermore, the expression of the lymphangiogenic factors in response to the modulation of EGFR signalling by the EGFR-targeted monoclonal antibody cetuximab was investigated at the mRNA and protein level in human SW480 and SW620 CRC cell lines and a mouse xenograft model. RESULTS: Human CRC specimens and cell lines displayed EGFR, VEGF-C and VEGF-D expression with varying intensities. VEGF-C expression was associated with histological grade. Strong expression of VEGF-D was significantly associated with lymph node metastases and linked to a trend for decreased survival in lymph node-positive patients. EGFR blockade with cetuximab resulted in a significant decrease of VEGF-D expression in vitro and in vivo. CONCLUSION:In conclusion, the expression of VEGF-D in colorectal tumours is significantly associated with lymphatic involvement in CRC patients and such expression might be blocked effectively by cetuximab.

Induced dural lymphangiogenesis facilities soluble amyloid-beta clearance from brain in a transgenic mouse model of Alzheimer's disease

Impaired amyloid-β clearance from the brain is a core pathological event in Alzheimer＇s disease.The therapeutic effect of current pharmacotherapies is unsatisfactory,and some treatments cause severe side effects.The meningeal lymphatic vessels might be a new route for amyloid-β clearance.This study investigated whether promoting dural lymphangiogenesis facilitated the clearance of amyloid-β from the brain.First,human lymphatic endothelial cells were treated with 100 ng/m L recombinant human vascular endothelial growth factor-C（rh VEGF-C） protein.Light microscopy verified that rh VEGF-C,a specific ligand for vascular endothelial growth factor receptor-3（VEGFR-3）,significantly promoted tube formation of human lymphatic endothelial cells in vitro.In an in vivo study,200 μg/m L rh VEGF-C was injected into the cisterna magna of APP/PS1 transgenic mice,once every 2 days,four times in total.Immunofluorescence staining demonstrated high levels of dural lymphangiogenesis in Alzheimer＇s disease mice.One week after rh VEGF-C administration,enzyme-linked immunosorbent assay results showed that levels of soluble amyloid-β were decreased in cerebrospinal fluid and brain.The Morris water maze test demonstrated that spatial cognition was restored.These results indicate that the upregulation of dural lymphangiogenesis facilities amyloid-β clearance from the brain of APP/PS1 mice,suggesting the potential of the VEGF-C/VEGFR-3 signaling pathway as a therapeutic target for Alzheimer＇s disease.

Impact of lymph node micrometastasis in hilar bile duct carcinoma patients

AIM- To immunohistochemicaUy examine micrometastasis and VEGF-C expression in hilar bile duct carcinoma （HBDC） and to evaluate the clinical significance of the results. METHODS： A total of 361 regional lymph nodes from 25 patients with node-negative HBDC were immunostained with an antibody against cytokeratins 8 and 18 （CAM 5.2）, and immunohistochemical staining of VEGF-C was performed in 34 primary resected tumors. RESULTS： Lymph node micrometastasis was detected in 6 （24%） of the 25 patients and 10 （2.8%） of the 361 lymph nodes. Patients with micrometastasis showed significantly poorer survival rates than those without （P= 0.025）. VEGF-C expression was positive in 17 （50%） of 34 HBDC, and significantly correlated with lymph node metastasis （P=0.042） and microscopic venous invasion （P=0.035）. CONCLUSIONS： It is suggested that immunohistochemically detected lymph node micrometastasis has an impact on the outcome of HBDC. VEGF-C expression is highly correlated with lymph node metastasis in HBDC and might therefore be a useful predictor.

Application and prospect of adipose stem cell transplantation in treating lymphedema

BACKGROUND Lymphedema is a chronic,debilitating and incurable disease that affects 0.13%-2%of the global population.Emerging evidence indicates that adipose-derived stem cells(ADSCs)might serve as suitable seed cells for lymphatic tissue engineering and lymphedema therapy.AIM To summarize applications of ADSCs for treating lymphedema in both animal studies and clinical trials.METHODS A systematic search was performed on four databases-PubMed,Clinicaltrials.gov,the evidence-based Cochrane Library,and OVID-using the following search string:(“lymphedema”or“lymphoedema”or“lymphangiogenesis”)and(“adipose-derived stem cells”or“adipose-derived stromal cells”or“adipose-derived regenerative cells”).A manual search was performed by skimming the references of relevant studies.Animal studies and clinical trials using adipose-derived cells for the treatment of any kind of lymphedema were included.RESULTS A total of eight research articles published before November 2019 were included for this analysis.Five articles focused on animal studies and another three focused on clinical trials.ADSC transplantation therapy was demonstrated to be effective against lymphedema in all studies.The animal studies found that coadministration of ADSCs and controlled-release vascular endothelial growth factor-C or platelet-rich plasma could improve the effectiveness of ADSC therapy.Three sequential clinical trials were conducted on breast cancer-related lymphedema patients,and all showed favorable results.CONCLUSION ADSC-based therapy is a promising option for treating lymphedema.Large-scale,multicenter randomized controlled trials are needed to develop more effective and durable therapeutic strategies.

Inhibition of lymphangiogenesis,nodal and lung metastasis by dihydroartemisinin in mice bearing Lewis lung carcinoma

Objective:To investigate the activity of anti-malarial dihydroartemisinin (DHA) on tumor growth, lymphangiogenesis, nodal and lung metastasis and survival in mice bearing Lewis lung carcimoma (LLC). Methods: The models of C57BL/6 mice transplantation tumors were established via subcutaneous injection of LLC cells and divided into 4 groups: control group, DHA group, DHA+ferrous sulfate (FS) group and FS group, with 25 mice in each group. Tumor volumes and weights, nodal and lung metastasis, and survival were monitored. Tumor lymphatic microvessel density (LMVD) was determined by lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1) immnohistochemistry. After LLC cells were treated with DHA or DHA+FS, protein and mRNA levels of vascular endothelial growth factor (VEGF) -C were evaluated by Western blotting and real time quantitative RT-PCR, respectively. Results: Oral administration of DHA or DHA+FS inhibited lymph node and lung metastasis, and prolonged survival. However, no significant tumor growth retardation effect was observed when mice were treated with DHA alone. The inhibited tumor metastasis was related to the decreased LMVD in the peritumoral regions, but not in the intratumoral regions. DHA significantly down-regulated the expression of VEGF-C protein and mRNA in LLC cells. Conclusion: DHA effectively inhibits LLC transplantation tumor lymphangiogenesis, nodal and lung metastasis, and may be a promising chemotherapeutic agent for controlling lung cancer metastasis by decreasing VEGF-C expression.

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.