Objective To investigate the role of vascular endothelial growth factor-C (VEGF-C) and its receptors in the formation of lymphatic vessels and lymphatic metastasis in gastric cancer. Methods By the domestic and overseas literatures review, the expressions of VEGF-C and its receptors in gastric cancer, their role in tumor lymphatic metastasis and prospect in treatment of gastric cancer were summarized.Results There was a significant correlation between VEGF-C and its receptors and the formation of lymphatic vessels and lymphatic metastasis in gastric cancer. VEGF-C high expression might be an early event in lymphatic metastasis and could be considered as an independent predictive factor of lymphaticmicrometastasis. By inhibition of gastric cancer cell from secrete VEGF-C or blockage of the interaction of VEGF-C with VEGFR3, it was possible to inhibit tumor angiogenesis and the invasion and distant spread of cancer cells, thereby decreased mortality and improve survival. ConclusionVEGF-C and its receptors may promote the formation of lymphatic vessels and lymphatic metastasis in gastric cancer. It may be an effective way to gastric cancer for the treatments against VEGF-C and its receptors.
ObjectiveTo investigate the relationship between vascular endothelial growth factor receptor-3 (VEGFR-3) and clinical pathology of gastric carcinoma(GC).MethodsThe expression of VEGFR-3 in 80 GCs and 20 gastric benign tissues (GBT) was detected by immunohistochemistry(SP), by which the density of lymphatic vessels (DLV) was calculated. ResultsThe DLV in GC was （5.800 0±2.318 9）/×200, in GBT （2.380 0±0.462 9）/×200(P=0.000); in GC with lymph node metastasis （6.948 3±1.583 1）/×200, without lymph node metastasis （2.772 7±0.428 9）/×200 （P=0.000）. In poorly differentiated type group, DLV was （7.681 8±0.982 9）/×200, higher than that in moderately and highly differentiated type group 〔(3.500 0±1.028 2）/×200, P=0.000〕. DLV in pTNM Ⅰ+Ⅱ was （4.291 7±1.688 0）/×200, in Ⅲ+Ⅳ （8.062 5±0.759 4）/×200 （P=0.000）.ConclusionDLV shows positive relations with pTNM stage, differentiation and lymph node metastasis of GC.
ObjectiveTo investigate the expressions of contactin-1 (CNTN-1), vascular endothelial growth factor-C (VEGF-C), and its receptor VEGFR-3 (Flt-4) in primary gastric cancer and to explore the relevance among them and their correlation with clinicopathologic features of gastric cancer. MethodsThe VEGF-C, VEGFR-3, and CNTN-1 protein expressions of tumor tissues and normal gastric mucosa tissues in 68 patients with primary gastric cancer were analyzed by immunohistochemistry. The Flt-4-positive vessel density (FVD) and lymphatic vessel density (LVD) were also analyzed by VEGFR-3positive and D2-40-positive staining, respectively. ResultsThe positivity rate of VEGF-C, VEGFR-3, and CNTN-1 protein expression in the primary tumor was 57.4% (39/68), 60.3% (41/68), and 55.9% (38/68), respectively, which was significantly higher than that in the normal gastric mucosa tissues 〔20.6% (14/68), 23.5% (16/68), and 16.2% (11/68)〕， P=0.000. The expressions of VEGF-C, VEGFR-3, and CNTN-1 protein were significantly correlated with TNM stage, lymphatic vessel invasion, and lymph node metastasis (Plt;0.05). The expression of CNTN-1 protein was significantly correlated with VEGF-C (r=0.372, P=0.002) and VEGFR-3 protein expression (r=0.308, P=0.011). In tumor tissues of sixtyeight patients the FVD was (10.41±9.38)/HP, which was significantly lower than LVD 〔(18.19±7.44)/HP〕, P=0.000. Elevated FVD and LVD was significantly found in patients with tumor characterized by later TNM stage, severer lymphatic vessel invasion, and severer lymph node metastasis (Plt;0.05). The FVD of tumor was significantly correlated with VEGF-C (P=0.029) and CNTN-1 protein expression (P=0.003). The LVD of tumor was not significantly correlated with CNTN-1 (P=0.727), VEGF-C (P=0.173), and VEGFR-3 protein expression (P=0.924). The patients with positive expression of VEGF-C, VEGFR-3, and CNTN-1 protein showed poorer prognosis (Plt;0.05). ConclusionsElevated expression of CNTN-1 protein is observed in primary gastric cancer and correlated with VEGF-C and VEGFR-3 protein expression, indicating that combined detection has great value in prediction of invasive potential and prognosis. VEGF-C-mediated CNTN-1 overexpression may promote lymphatic invasion via lymphangiogenesis pathway in patients with gastric cancer.
Objective To compare the transfection effects on soluble fms-like tyrosine kinase receptor-1 (sFlt-1) gene (2-4 transcellular region) mediated by carboxymethylated dextran coated nanoparticle and lipofectamineTM2000.Methods The plasmid pcDNA3.1-EGFP/sFlt-1(2-4) was constructed and assessed by enzyme cut, electrophoresis, and genetic sequencing. Three groups were divided: nanoparticle group, lipofectamine group, and non-transfected group. Twenty-four and 48 hours after the transfection, the distribution of cellular green fluorescence was oberved under the inverted phase contrast fluorescence microscope; the expression rate of green fluorescence was measured by flow cytometry; the expression of sFlt-1(2-4)mRNA and the protein was detected by reverse transcriptionpolymerase chain reaction (RT-PCR) and Western blot; the growth of the cells was observed by methyl thiazolyl tetrazolium (MTT) colorimetry and the relative growth rate (RGR) of the cells in each group was calculated; the cellular apoptosis in each group was detected by Hoechst staining.Results The sequence of sFlt-1(2-4) gene was equal to 915 base pair (bp).The transfection rate was 45% in nanoparticle group and 21% in lipofectamine group; the difference between the two groups was significant (t=2.541，Plt;0.05). Forty-eight hours after the transfection, the expression of sFlt-1（2-4）mRNA and protein was obviously higher in nanoparticle group than that in lipofectamine group (t=2.454，2.398；Plt;0.05) . Twenty-four and 48 hours after the transfection,the difference of RGR of the cells between nanoparticle and non-transfected group was not significant（t=1.436，Pgt;0.05）; the RGR in lipofectamine group differed much from that in non-transfected and nanoparticle group (t=2.412，2.545; Plt;0.05) ; the difference of cellular apoptosis was not significant between nanoparticle and nontransfected group (t=1.436，Pgt;0.05), but significant between nanoparticle and lipofectamine group (t=2.236，Plt;0.05). Conclusion The transfection rate of sFlt-1(2-4) mediated by carboxymethylated dextran coated nanoparticle was higher than that mediated by lipofectamineTM2000.
Objective To observe the expression of vascular endothelial growth factor receptor-1 (VEGFR-1) and VEGFR-2 in hypoxic chorioretinal endothelial cells of monkeys (RF/6A), and to evaluate the effect of minocycline. Methods RF/6A was cultured and divided into four groups: control group, hypoxia group, hypoxia and low dose of minocycline group (0.5 mu;mol/L), hypoxia and medium dose of minocycline group (5 mu;mol/L), and hypoxia and high dose of minocycline group (50 mu;mol/L). Real-time reverse transcriptionpolymerase chain reaction (RT-PCR) and immunohistopathological staining were used to measure the mRNA and protein expression of VEGFR-1 and VEGFR-2, respectively. Results RT-PCR showed that the expression of VEGFR-1 mRNA did not vary significantly between groups (F 24 h=0.17,F 48 h=1.53,F72 h=2.04;P＞0.05). Compared with hypoxia group, the expression of VEGFR-2 mRNA in all minocycline treated groups were significantly downregulated (low minocycline, medium minocycline, high minocycline: t=4.69, 20.16, 17.12; P＜0.001). The immunohistopathological study showed the cells with positive staining of VEGFR-1 can be observed in all groups, and the staining was relatively weak and mainly located in cell membrane and cytoplasm. The optical density value analysis showed that the protein expression of VEGFR-1 did not vary significantly between groups at all time points（F 24 h=0.251,F 48 h=0.340,F72 h=0.589;P＞0.05）. The VEGFR-2 positive staining cells were also observed in all groups, and the staining was relatively high. Brown staining particles of VEGFR-2 were observed in the cell membrane with minor staining particles in cytoplasm. The staining density of VEGFR-2 was significantly higher in hypoxia group than control group. Compared with the hypoxia group, the protein expression of VEGFR-2 in minocycline treated groups was significantly lower（F 24 h=19.147,F 48 h=14.893,F72 h==11.984; P＜0.05). Conclusion The expression of VEGFR-2 is upregulated in RF/6A, and minocycline somewhat shows an inhibition effect.
Objective To explore and evaluate the protective effects of soluble fms-like tyrosine kinase recptor-1(sFlt-1) gene 2-3 and 2-4 transcellular region on retinal vascular leakage and phosphatidyl inositol-3 kinase (PI3K)/Akt pathway under hypoxia and/or hyperglycemia. Methods The plasmids pcDNA3.1-EGFP/sFlt-1(2-3) and pcDNA3.1-EGFP/sFlt-1(2-4) were constructed.. Two of these plasmids with carboxy methylation glucan (CMD) magnetic particles were transfected into human umbilical vein endothelial cells (HUVEC), which were cultured under hypoxia and/or hyperglycemia. The blood retinal barrier was evaluated by Evans blue permeation (EBP). Then the expression of p-Akt mRNA and protein were detected by reverse transcriptionpolymerase chain reaction (RT-PCR) and Western blot separately. Results In the diabetic rabbits, The blood retinal barrier breakdown was detected by the retinal vascular leakage of EBP. The expression of p-Akt mRNA and protein in hypoxia and hyperglycemia groups were also obviously increased. These changes were largely prevented by transfection the plasmids pcDNA3.1-EGFP/sFlt-1(2-3) and pcDNA3.1-EGFP/sFlt-1(2-4) (P＜0.01 in both groups). Conclusion Both sFlt-1(2-3) and sFlt-1(2-4) can make the retinal blood vessels less leaky and may be beneficial in preventing vision loss from diabetic retinopathy.
【Abstract】Objective To understand the features of lymphatic vessel, and to summarize the foundation and mechanism of the promotion and inhibition of tumor lymphangiogenesis recorded on the current studies of animal experiments and clinical researches. Methods The related literatures of the structural features of lymphatic vessel, lymphatic endothelial molecular markers, the origin of lymphatic tumors, the molecular mechanisms and regulatory factors were reviewed, and the relationship between tumor lymphangiogenesis and lymphatic metastasis, the treatment targeting at the formation of the anti-tumor lymphatic vessel and its existing problems were also analyzed. Results Hyperplasia of lymphatic vessels occurred during the process of tumor formation and progression. The structural features of the lymphatic vessels in the tumor were conducive to tumor lymphatic metastasis. In recent years, methods of anti-lymphangiogenesis and inhibition of tumor lymphatic metastasis had achieved considerable success in animal experiments. However, there were still a lot of problems need to be solved. Conclusion Tumor lymphangiogenesis has a significantly positive correlation between tumor lymphatic metastasis and patients’ prognosis, which may indicate that treatment against the formation of tumor lymphatic vessel maybe effective.
ObjectiveTo observe the stoichiometry of vascular endothelial growth factor receptor 2 (VEGFR2) on the retinal vascular endothelial cell membrane by single-molecule fluorescence imaging.MethodsRhesus monkey retinal vascular endothelial cells (RF/6A) were divided into blank control group (normal culture) and plasmid transfection group [transfected with VEGFR2-green fluorescent protein (GFP) recombinant plasmid]. The expression of GFP in the plasmid transfected group was observed by confocal microscope, and the expression of VEGFR2 in the cells was detected by real-time fluorescent quantitative polymerase chain reaction (qPCR) and Western blot. The fluorescence intensity distribution and bleaching steps of single VEGFR2-GFP molecule on the cell membrane were recorded by single-molecule imaging. The distribution of fluorescence intensity and the number of fluorescence bleaching steps of GFP were recorded.ResultsGFP green fluorescence was observed in the transfected cells 12 hours after transfection. qPCR results showed that the expression of VEGFR2 and GFP mRNA in the plasmid transfected group was significantly higher than that in the blank control group (t=11.240, 12.330; P＜0.001, 0.001). Western blot results showed that the expression of VEGFR2 protein in the plasmid transfected group was significantly higher than that in the blank control group (t=8.346, P＜0.01). The results of single-molecule imaging showed that the fluorescence intensity distribution of VEGFR2-GFP on the surface of RF/6A cell membrane without ligand stimulation was bimodal, in which monomer and dimer were 86.0% and 14.0% respectively. By counting the steps of GFP fluorescence bleaching, the proportions of receptor monomer, dimer, trimer, and tetramer were 81.4%, 12.9%, 5.5%, and 0.3% respectively.ConclusionIn the absence of ligands, VEGFR2 coexists in the form of monomers and dimers on the surface of RF/6A cell membrane, and monomers are dominant.
Objective o observe the expression of Notch1 and Delta-like ligand 4 (Dll4) on the fibrovascular membranes in proliferative diabetic retinopathy (PDR), and investigate its relationship with vascular endothelial growth factor receptor 2 (VEGFR2). Methods Fifty-seven PDR patients (60 eyes) who underwent vitrectomy were enrolled in this study. The PDR patients were divided into non-injection group (30 patients, 32 eyes) and injection group (27 patients, 28 eyes). The eyes in injection group received intravitreal injection with ranibizumab at 2 to 7 days before surgery. The preretinal fibrovascular membranes were obtained from the PDR patients during vitrectomy. Eighteen epiretinal membranes were obtained from the non-diabetic patients was served as controls. The real-time polymerase chain reaction (RT-PCR) and immunohistochemical methods were used to detecting the expression of Notch1, Dll4 and VEGFR2. In the meantime, the numbers of the nucleus of vascular endothelial cells in the membranes stained with hematoxylin were counted. Results The immunohistochemical staining revealed that there were positive expression of Notch1, Dll4 and VEGFR2 in all PDR membranes, regardless of the injection of the ranibizumab. The levels of Notch1, Dll4 and VEGFR2 protein in non-injection group were higher than those of injection group (t=3.45, 6.01, 4.08;P=0.030, 0.008, 0.023). In injection group, the number of endothelial cells in the membranes reduced (17.17±2.48) compared with that of the non-injection group (41.50±5.57). There was significant difference in the number of endothelial cells in the membranes between the two groups (t=9.58,P＜0.05). RT-PCR showed that the differences of the mRNA expression of Notch1, Dll4 and VEGFR2 were all statistically significant among the PDR group and control group (H=12.50, 12.50, 12.02;P＜0.05).The expression of Notch1, Dll4 and VEGFR2 in the PDR membranes was higher than that of epiretinal membranes from non-diabetic patients. In the PDR group, the expression of Notch1, Dll4 and VEGFR2 of non-injection group was higher than that of injection group. Spearman correlation analysis showed that the expression of mRNA between VEGFR2 and Dll4 (r=0.83), VEGFR2 and Notch1 (r=0.81), Notch1 and Dll4 (r=0.87) were all significantly correlated (P＜0.05). Conclusions The expression of Notch1 and Dll4 in the PDR membranes are higher than that of the control group, and it is positively correlated with the expression of the VEGFR2. Notch1 and Dll4 play a regulatory rule in the neovascularization in PDR, the acting way may be correlated with VEGFR2.
Objective To explore the effects of SU5416，a vascular endothelial growth factor receptor (VEGFR) inhibitor，on inflammation in mice with bleomycin-induced pulmonary fibrosis.Methods C57BL/6 mice were randomly divided into four groups，ie.a control group，a bleomycin-induced pulmonary fibrosis (BPF) group,early intervention with SU5416 group (days 1-14，SE)，and delayed intervention with SU5416group (days 15-28，SD).Bleomycin (10 mg/kg) was instilled into the trachea of the mice to induce pulmonary fibrosis.After the instillation，SU5416 (50 mg/kg) was injected into the peritoneal cavities of the mice twice a week.The mice treated with bleomycin alone or with bleomycin followed by SU5416 were sacrificed on the day 14 and day 28 after the instillation of bleomycin.Lung tissues were taken and processed for determination of hydroxyl proline content.Bronchoalveolar lavage fluid (BALF) was collected for total and differential cell counts and measurements of lactic dehydrogenase (LDH) levels.Results It was found that the hydroxyl proline contents，the number of macrophages，and the LDH contents from the mice treated with bleomycin alone on day 14 were greatly increased.However，only the hydroxyl proline showed a significant increase in the mice treated with bleomycin alone on day 28.The number of macrophages，LDH contents，and hydroxyl proline contents were greatly reduced in the mice treated with bleomycin and early administration of SU5416 on day 14 when compared with those treated with bleomycin alone.But there were no significant differences in the above parameters of the mice treated with bleomycin and delayed administration of SU5416 on day 14.Conclusion This study indicates that early intervention by SU5416 might attenuate the fibrosis through inhibiting early inflammation in animal model of pulmonary fibrosis induced by bleomycin.