Vascular endothelial growth factor expression in superficial transitional cell bladder carcinoma

AIM:To study the expression of the VEGF in single and multiple superficial transitional cell bladder carcinoma and their clinical significance.METHODS:Immunohistochemical method was used to study the VEGF in 60 cases of superficial transitional cell bladder carcinoma and in 10 cases of normal bladder tissue as control. RESULTS:High expression of VEGF in bladder carcinoma cell was observed. The expression level of VEGF in multiple superficial transitional cell bladder carcinoma was higher than that in single superficial transitional cell bladder carcinoma. The recurrent rate in the patient with VEGF high expression was more than that in the patient with VEGF low expression. CONCLUSION:The expression level of VEGF was correlated to the biological behavior of superficial transitional cell bladder carcinoma.     

Microvessel density and expression of vascular endothelial growth factor in glomeruli of diabetic mice

AIM: To investigate the relationship between microvessel density (MVD) and expression of vascular endothelial growth factor (VEGF) in glomeruli of diabetic mice. METHODS: Streptozotocin-induced diabetic mice as well as the control mice were involved in this study for 6 weeks. The body weight and blood glucose level of the mice in each group were weekly measured at certain time point. The morphological changes of the kidney were observed under light microscope, and the diameter, perimeter and area of the glomeruli were detected by an image analysis system. The expression of CD34 and VEGF in glomeruli was examined by immunohistochemistry method, and MVD and VEGF index were also calculated. RESULTS: In comparison with the control mice, the blood glucose level was significantly increased,and the body weight was decreased in diabetic mice(P<0.01). The diameter, perimeter and area of glomeruli in diabetic mice were significant greater than those in control mice (P<0.05). Increased expression of CD34 and VEGF in the glomeruli of diabetic mice was observed. Glomerular MVD of diabetic mice was significantly higher than that of the controls (P<0.01), and was positively correlated with the VEGF index (r=0.9979, P<0.05). CONCLUSION: VEGF may promote the angiogenesis in glomeruli of diabetic mice. The increase in VEGF expression may play a role in the pathogenesis of diabetic nephropathy.     

Effects of conditioned medium of ADSCs transfected with VEGF on dermal fibroblasts and umbilical vein endothelial cells in vitro

AIM: To investigate the influence of conditioned medium (CM) of human adipose-derived stem cells (HADSCs) transfected with vascular endothelial growth factor (VEGF) gene on human dermal fibroblasts (HDFs) and human umbilical vein endothelial cells (HUVECs) in vitro.METHODS: The HADSCs, HDFs and HUVECs were prepared and identified. The HADSCs were transfected with lentivirus carrying VEGF165 gene and the CM was collected regularly. ELISA method was used to detect the growth factor secretion in the CM. The VEGF-CM mixed with complete medium were divided into 5 groups for culturing with HDFs or HUVECs. The cell viability was measured by CCK-8 assay. The optimal ratio of VEGF-CM, normal CM (Nor-CM) and complete medium were applied to HDFs or HUVECs. The migration ability was detected by wound-healing assay. RESULTS: The results of ELISA showed that the expression levels of VEGF and basic fibroblast growth factor (bFGF) in VEGF-CM group were higher those in Nor-CM group (P<0.05). Compared with other groups, the cell viability and migration abilities of HDFs and HUVECs were obviously enhanced in VEGF-CM group (P<0.05). CONCLUSION: The HADSCs transfected with VEGF165 gene greatly enhances the expression of VEGF and bFGF. The CM of HADSCs promotes the viability and migration abilities of HDFs and HUVECs in vitro.     

Role of focal adhsion kinase in vascular endothelial cell migration

Focal adhesion kinase (FAK) is a non-receptor protein tyrosine kinase (PTK) that can localize indirectly to sites of clustering integrin family of heterodimeric receptors. As an important structure and signaling molecule in the adhesive complexes, which are large and stable referred as 'focal adhesions’ or relatively small and transient within filopodia and lamellipodia named 'focal complexes’, FAK is closely related with cell death, proliferation and migration. In this review, we discuss the function of FAK in the regulation of endothelial cell migration based on current data.     

Ligustilide inhibits angiogenesis and epithelial-mesenchymal transition in human hemangioendothelial cells by reducing VEGF levels

AIM To investigate the effect of ligustilide on human hemangioendothelial cells (HemECs) and to analyze its mechanism. METHODS The effect of ligustilide at different concentrations on the viability of HemECs was measured by CCK-8 assay. The HemECs were divided into control group and ligustilide （10, 25 and 50 μmol/L） treatment groups, and the proliferation of HemECs was detected by EdU staining. The effects of ligustilide on the angiogenesis of HemECs was tested by microtubule formation experiment. The protein expression of vascular endothelial growth factor (VEGF) and epithelial-mesenchymal transition (EMT)-related markers in HemECs cells was determined by Western blot. RESULTS There was no significant difference in the viability of the cells treated with ligustilide at the concentrations between 0.1~50 μmol/L compared with control cells. Compared with control group, ligustilide at 25 and 50 μmol/L significantly reduced the number of EdU-positive cells and microtubule-like structures (P<0.05), reduced the protein expression level of VEGF (P<0.05), increased the protein expression of E-cadherin, and decreased the protein expression of vimentin and β-catenin (P<0.05). Compared with control group, the expression of VEGF and vimentin was significantly up-regulated, and the protein expression of E-cadherin was significantly down-regulated in VEGF overexpression group (P<0.05). Compared with VEGF overexpression group, the expression of VEGF and vimentin in 50 μmol/L ligustilide-treated VEGF-overexpressing cells were significantly reduced (P<0.05), and the protein expression of E-cadherin was significantly increased (P<0.05). CONCLUSION Ligulide inhibits the proliferation of HemECs, and also inhibits the angiogenesis and EMT process of HemECs by reducing the level of VEGF.     

Establishing a three-dimensional angiogenesis model in vitro and secretion of MCP-1 and VEGF by tumor cells

AIM: To establish a three-dimensional angiogenesis model in vitro for observing the influence of tumor cells on angiogenesis, and to explore its possible molecular mechanism. METHODS: Human umbilical vein endothelial cells (HUVECs) and mouse endothelial cells SVEC4-10EE2 were coated on the surface of beads, and then mixed with fibrinogen and seeded in cell culture plates containing thrombin. The cultured endothelial cells coated on the beads grew into a vessel-like structure in a three-dimensional space containing fibrin condensate to establish an in vitro three-dimensional angiogenesis model. Tumor cells MDA-MB-231 and E0771 were co-cultured in the model to observe the effect of tumor cells on angiogenesis in vitro,respectively. The concentrations of monocyte chemoattractant protein-1 (MCP-1) and vascular endothelial growth factor (VEGF) in the tumor cells culture supernatant were measured by ELISA. An antagonist of CCR2 (receptor of MCP-1), along with SU5416 (an inhibitor of VEGF receptor), were added to the tumor cell co-culture system. The supernatant of the tumor cells was collected as a conditioned medium, which was then added to the angiogenesis system of HUVECs or SVEC4-10EE2 cultured individually. The effect of conditioned medium on angiogenesis was observed under the conditions of with or without SU5416, as well as with or without CCR2 antagonist. RESULTS: Under normal condition, HUVECs and SVEC4-10EE2 formed a multi-cellular vascular structure the three-dimensional angiogenesis model in vitro. The co-culture of MDA-MB-231 (E0771) cells significantly promoted in the formation of membrane-like structures. The ELISA results showed that the levels of MCP-1 and VEGF in the supernatant of tumor cells were significantly elevated (P<0.05). MCP-1 promoted the formation of membrane tube in three-dimensional culture system in vitro. After adding the antagonists of MCP-1 receptor CCR2 and VEGF (SU5416), the angiogenesis was significantly inhibited (P<0.05). At the same time, conditioned medium promoted the formation of extracorporeal membranes in the endothelial cells. The promoting effect was blocked by CCR2 antagonists and SU5416 (P<0.05). CONCLUSION: The in vitro three-dimensional angiogenesis model is successfully established. Tumor cells significantly promote the formation of membrane tubes. The effect of tumor cells might be related to MCP-1 and VEGF secretion.     

Promoting angiogenesis of protein kinase D1 in vitro and in vivo

AIM: To explore the effect of protein kinase D1 (PKD1) on promoting angiogenesis in vitro and in vivo, and to furnish a new idea on targeting PKD1 for the treatment of ischemic heart disease such as myocardial infarction. METHODS: The culture, isolation and identification of endothelial progenitor cells (EPCs) were performed in vitro. The effects of PKD1 and its specific blocking agent CID755673 on expression levels of vascular endothelial growth factor (VEGF) and kinase insert domain receptor (KDR) in EPCs were determined. The rat model of myocardial infarction was established, the intervention effects of PKD1 and CID755673 on morphology, changes of microvessels and endothelial cells, and the expression of VEGF and KDR in the impaired myocardial tissue were analyzed. RESULTS: PKD1 significantly upregulated the expression of VEGF and KDR in EPCs in vitro. Meanwhile, the structure of myocardial tissue was more regular and clear, the cytomembrane of endothelial cells were more smooth and integrity, the pericytes were visible, and the expression of VEGF and KDR was significantly increased in PKD1 treatment group in vivo.CONCLUSION: PKD1 has the ability of angiogenesis obviously, which might be mediated by VEGF.     

Effects of cellular repressor of E1A-stimulated genes on VEGF release and monolayer permeability of human vascular endothelial cells

AIM: To study the effects and mechanism of cellular repressor of E1A-stimulated genes (CREG) on VEGF release and monolayer permeability of human vascular endothelial cells (ECs). METHODS: The monolayer permeability of ECs was measured by transwell chamber model. The expression and localization of F-actin and VE-cadherin were examined by immunofluroscence using Olympus IX-70 fluorescent microscope. Enzyme-linked immunosorbent assay (ELISA) were performed to determine the concentration of vascular endothelial growth factors(VEGF) in the culture medium. VEGF neutrilization antibody was used to block the expression of VEGF in the cells. RESULTS: The monolayer permeability of CREG over-expressing ECs (EO group) was significantly higher than that of the normal control ECs (EN group, P<0.05). The monolayer permeability of CREG suppressing ECs (ES group) was lower than that in EN group (P<0.05). F-actin cytoskeleton in EO group showed disorganized, polymerized and bundled obviously to form large quantity of stress fibers in the central portion of the cells, whereas F-actin in EO group was mainly observed in the peripheral portion of the cells and only small amounts in the central portion of the cells. A widespread gap formation and a loss of VE-cadherin staining at the periphery were found in the cells of EO group. Inversely, the cells in ES group showed the localization of VE-cadherin at the cell-cell contacts tightly and the formation of zipper-like structures. Compared with EN group, the secretion of VEGF in the cell culture supernatants increased in EO group, but decreased in ES group (P<0.05). Furthermore, the changes of ECs permeability, cytoskeleton reorganization and loss of VE-cadherin induced by CREG were abolished by the addition of anti-VEGF neutralizing antibody. CONCLUSION: CREG over-expression increases the monolayer permeability of ECs, induces the cytoskeleton reorganization and reduces VE-cadherin expression by enhancing the secretion of VEGF in vitro.     

Inhibition of K562 cell growth by antisense drug targeting with VEGF mRNA in vitro

AIM: To investigate inhibition of K562 cell growth by antisense drug targeted VEGF mRNA. METHODS: X7, 20-mer antisense sequences were selected, synthesized and modified with phosphorothioate. The drug was transfected into K562 cells in the present of lipofection. Cell growth was assayed by trypan blue dye exclusion assay and MTT. The level of VEGF protein in the media was determined by ELISA. The morphology of apoptotic cells were observed by Giemsa staining, and the propotion of apoptotic cells was detected by flow cytometry. RESULTS: The antisense drug inhibited growth of K562 and downregulated expression of VEGF protein significantly, compared with Scrambed control group and showed dose-dependent relation. Signs of apoptosis of K562 cells were not observed. CONCLUSION: Inhibition of K562 cell proliferation, but not cells apoptosis induction is the mechanism of inhibing growth of K562 cells by antisense drug targeted VEGF mRNA. At same time, VEGF has function of promoting K562 cell proliferation, and VEGF mRNA may be a new target attached by drugs.     

Genistein inhibits proliferation of human oral cancer TCA8113 cells through suppression of VEGF expression

AIM: To investigate the effect of genistein on the proliferation of human oral cancer TCA8113 cells and to explore the underlying mechanisms.METHODS: The cell proliferation was examined by MTT assay, cell counting and colony formation assay. Western blotting was employed to examine the protein levels of vascular endothelial growth factor(VEGF), extracellular signal-regulated kinase(ERK) and p-ERK. RESULTS: Genistein significantly inhibited the proliferation of TCA8113 cells in a concentration-dependent fashion. Moreover, genistein dose-dependently decreased the protein levels of VEGF, ERK and p-ERK. The expression of VEGF was also blunted by U0126, a specific inhibitor of ERK. U0126 and axitinib, a VEGF receptor antagonist, both significantly inhibited the proliferation of TCA8113 cells. CONCLUSION: Genistein inhibits the proliferation of TCA8113 cells, which may be related to its inhibitory effect on ERK expression and activation, thus subsequently decreasing the expression of VEGF.     

Effects of phillyrin on VEGF and endostatin expression in Lewis lung carcinoma

AIM: To investigate the effects of phillyrin on vascular endothelial growth factor (VEGF) and endostatin expression in lung tumor tissues isolated from Lewis lung carcinoma. METHODS: The expression of VEGF and endostatin in control individuals and the patients with lung cancer was determined by immunohistochemistry. In the animal experiment, 5 groups of animals were examined: control, tumor model, and tumor model with 3 different concentrations of phillyrin treatments. For preparation of transplanted tumor model, Lewis cells were subcutaneously injected into the right limb armpit of the nude mice. After that, phillyrin was administered via oral gavage once daily for 20 d at dose of 5 or 10 g/kg, or twice daily at 10 g/kg. Lung tumor tissues isolated from each group were observed by hematoxylin-eosin staining. VEGF and endostatin expression were examined by immunohistochemistry. RESULTS: VEGF expression was increased in lung tumor tissues as compared with normal and pericarcinous tissues, while endostatin expression was decreased. Phillyrin significantly inhibited the tumor size and tumor tissue density dose-dependently, which was accompanied with a decrease in VEGF expression and an increase in endostatin expression. CONCLUSION: Phillyrin inhibits the development of lung tumor through reducing VEGF expression and increasing endostatin expression.     

Expression of vascular endothelial growth factor in chronic inflammatory mucosa of lacrimal sac

AIM: To study the expression of vascular endothelial growth factor (VEGF) in inflammatory mucosa of lacrimal sac. METHODS: Immunohistochemical S-P method was used to examine the expression of VEGF in the mocusa from 12 patients with chronic dacryocystitis and 8 volunteers. RESULTS: The positive rates of VEGF expression in different parts of the mocusa were: basal lamina: 44.3%±7.6％; surface epithelium: 16.9%±4.6％; connective tissue: 15.2%±4.9％, all normal mocusa of 8 cases were negative. There was a significant difference between the two groups (P<0.01), a significant difference among each part of the chronic inflammatory mocusa of lacrimal sac. CONCLUSION: VEGF may play an important role in hyperplasia of inflammatory mucosa of lacrimal sac.     

Advances on studies of miR-21 in vascular endothelial function and angiogenesis

MicroRNAs (miRNAs)are a class of non-coding, endogenous, single-stranded small RNA molecules composed of 19~25 nucleotides. miRNAs are widely involved in the process of human life activities. Recent studies have shown that part of miRNAs regulate the vascular endothelial function and angiogenesis. High expression of miRNA-21 is found to play important roles in the cell proliferation, cell apoptosis, cell growth and death of vascular endothelial cells. This review will focus on the recent progress related to miRNAs in vascular endothelial function and angiogenesis, providing a new insight in cardiovascular disease prevention, clinical diagnosis, prognosis and target therapeutics.     

Effect of VEGF on proliferation and differentiation of neural stem cells from the hippocampal gyrus of rat in vitro

AIM: To observe the effects of vascular endothelial growth factor (VEGF) on the proliferation and differentiation of neural stem cells (NSCs) of rats in vitro.METHODS: NSCs isolated from the hippocampal gyrus of SD rats were primary cultured and subcultured,and then divided into two groups: (1) the cells in VEGF group were treated with 150 μg/L VEGF in the culture system,and VEGF was removed at the 7 th day;(2) control group (without VEGF treatment).The cellular morphology of two groups was observed by contrast phase microscope.Nestin and NF-200 expressing cells were detected via immunofluorescence method.The percentages of the immunostaining positive cells in each group at the 7 th day and at the 11 th day were determined.RESULTS: At the 7 th day,the percentage of nestin positive cells in VEGF group was 52.19%±7.95%,vs 29.26%±4.12% in control group (P<0.01).The percentage of NF positive cells in VEGF group was 22.33%±4.13%,vs 38.62%±5.31% in control group (P<0.01).At the 3 th day after VEGF was removed,the percentage of NF positive cells in VEGF group was 43.10%±3.70%,vs 30.56%±4.16% in control group (P<0.01).CONCLUSION: VEGF stimulates the proliferation of neural stem cells and inhibits their differentiation.     

Effect of water extract propolis on expression of vascular endothelial cell adhesion molecules

AIM: To explore the mechanism of propolis on the inhibition of atherosclerosis and thrombosis in injured human umbilical vascular endothelial cells (HUVECs) induced by tumor necrosis factor alpha (TNF-α)in vitro.METHODS: TNF-α at the concentration of 50 μg/L was used to induce the injury of HUVECs. The injured HUVECs were treated with water extract propolis (WEP) at the concentrations of 50, 100 and 200 mg/L for 6 h, 12 h and 24 h. The expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) was examined by flow cytometry.RESULTS: The expression of ICAM-1 and VCAM-1 was significantly higher in injured HUVECs (P<0.01) than that in the control cells. The expression of ICAM-1 and VCAM-1 was downregulated by WEP treatment in a dose-dependent manner. Between the groups of 100 and 200 mg/L WEP, the difference was significant. In the injured HUVECs treated with 50 mg/L WEP, the inhibitory effect on the expression of ICAM-1 and VCAM-1 was presented in a time-dependent manner. Compared to the single administration, the use of WEP combined with fluvastatin showed better inhibitory effect on the expression of ICAM-1 and VCAM-1 in the injured HUVECs induced by TNF-α (P<0.01).CONCLUSION: WEP may be helpful for the protection of vascular endothelial cells by inhibiting the expression of ICAM-1 and VCAM-1 in a time-and dose-dependent manner. The protective effect of WEP on endothelial cells may be synergic with the inhibitor of HMG-CoA reductase such as fluvastatin sodium.     

Effect of VEGF/bFGF complex peptide vaccine on toxicity and reproductivity in female-mice

AIM: To investigate the toxicity and reproductive effect of vascular endothelial growth factor (VEGF)/basic fibroblast growth factor (bFGF) complex peptide vaccine (VBP3) on the female-mice. METHODS: The VBP3 was purified with Ni-NTA affinity chromatography. The female BALB/c mice were immunized with the purified VBP3. The antibody titer in the serum was detected by ELISA. The data of the body weight and the organ weight of the parent mice were gathered and analyzed, and the pathological changes of the organs were observed with HE staining to investigate the toxicity of VBP3. To investigate the toxicity of VBP3 in the F1 mice, the parent immunized female mice were mated with the parent non-immunized male mice. After the F1 mice were born, the survival rate was calculated, the change of the body weight and the organ weight of the F1 mice were also determined. The pathological changes of the organs in F1 mice were also observed with HE staining. RESULTS: In the parent mice, high titers of the antibodies were detected against VEGF and bFGF, and no difference of the body weight, the organ weight and the pathological change between the immunized mice and control mice was found. In the F1 mice, a low titer of anti-bFGF antibody was detected compared with blank group. The survival rate in control group was higher than that in immunized group. In the 2 groups of the F1 mice, no obvious difference of the weight of the spleen, kidney, lung and heart was observed, and there was some difference in the weight of liver between the 2 groups. The results of the HE staining in the F1 mice showed some difference in the liver between the 2 groups. CONCLUSION: VEGF/bFGF complex peptide vaccine has no obvious organ toxicity in the parent female mice, but has some side effects on the reproductive and the healthy processes of F1 mice.     

VEGF promotes biliary epithelial cell viability and cystic dilation in rats with polycystic kidney

AIM:To explore the promoting effect of vascular endothelial growth factor (VEGF) on the viability of biliary epithelial cells and biliary cystic dilation in rats with polycystic kidney (PCK). METHODS:Immunohistoche-mical staining was used to detect the expression of VEGF (n=6) and CD31 (n=10) in the liver tissue of normal and PCK rats. RT-qPCR and ELISA were used to evaluate the expression levels of VEGF in rat biliary epithelial cells and culture supernatant. WST-1 assay was applied to measure the effect of VEGF on the viability of rat biliary epithelial cells, and the influence of cholangiocyte culture supernatant on the viability of rat vascular endothelial cells. The cell migration assay was employed to observe the effect of cholangiocyte culture supernatant on endothelial cell migration. Tube formation assay was used to assess the impact of cholangiocyte culture supernatant on the angiogenic ability of endothelial cells. RESULTS:The result of immunohistochemical staining manifested that VEGF was highly expressed in the cholangiocytes of the PCK rats (P<0.01). More newly formed blood vessels were observed in the hepatic portal area of PCK rats than that in normal rats (P<0.01). The results of RT-qPCR and ELISA suggested that the mRNA and protein expression levels of VEGF in the cholangiocytes of PCK rats were significantly higher than those in normal rats (P<0.01). VEGF enhanced the viability of cholangiocytes in PCK rats (P<0.01). The culture supernatant of cholangiocytes in PCK rats increased the endothelial cell viability (P<0.01). VEGF siRNA and VEGF receptor inhibitor reduced the viability of cholangiocytes (P<0.01). The results of cell migration assay and tube formation assay indicated that the abilities of endothelial cell migration and tube formation were improved by the culture supernatant of cholangiocytes in PCK rats (P<0.01). CONCLUSION:The bile duct cystic dilation of PCK rats was related to the excessive secretion of VEGF in bile duct epithelial cells.