Telencephalic stab wound injury induces regenerative angiogenesis and neurogenesis in zebrafish:unveiling the role of vascular endothelial growth factor signaling and microglia

After brain damage,regenerative angiogenesis and neurogenesis have been shown to occur simultaneously in mammals,suggesting a close link between these processes.However,the mechanisms by which these processes interact are not well understood.In this work,we aimed to study the correlation between angiogenesis and neurogenesis after a telencephalic stab wound injury.To this end,we used zebrafish as a relevant model of neuroplasticity and brain repair mechanisms.First,using the Tg(fli1:EGFP×mpeg1.1:mCherry)zebrafish line,which enables visualization of blood vessels and microglia respectively,we analyzed regenerative angiogenesis from 1 to 21 days post-lesion.In parallel,we monitored brain cell proliferation in neurogenic niches localized in the ventricular zone by using immunohistochemistry.We found that after brain damage,the blood vessel area and width as well as expression of the fli1 transgene and vascular endothelial growth factor(vegfaa and vegfbb)were increased.At the same time,neural stem cell proliferation was also increased,peaking between 3 and 5 days post-lesion in a manner similar to angiogenesis,along with the recruitment of microglia.Then,through pharmacological manipulation by injecting an anti-angiogenic drug(Tivozanib)or Vegf at the lesion site,we demonstrated that blocking or activating Vegf signaling modulated both angiogenic and neurogenic processes,as well as microglial recruitment.Finally,we showed that inhibition of microglia by clodronate-containing liposome injection or dexamethasone treatment impairs regenerative neurogenesis,as previously described,as well as injury-induced angiogenesis.In conclusion,we have described regenerative angiogenesis in zebrafish for the first time and have highlighted the role of inflammation in this process.In addition,we have shown that both angiogenesis and neurogenesis are involved in brain repair and that microglia and inflammation-dependent mechanisms activated by Vegf signaling are important contributors to these processes.This study paves the way for a better understanding of the effect of Vegf on microglia and for studies aimed at promoting angiogenesis to improve brain plasticity after brain injury.

Expression of vascular endothelial growth factor and its role in oncogenesis of human gastric carcinoma

AIM: To establish the role of vascular endothelial growth factor (VEGF) in the oncogenesis of human gastric carcinoma more directly. METHODS: The expression of VEGF and its receptor kinase-domain insert containing receptor (KDR) in human gastric cancer tissue were observed by immunohistochemical staining. VEGF levels were manipulated in human gastric cancer cell using eukaryotic expression constructs designed to express the complete VEGF(165) complimentary DNA in either the sense or antisense orientation. The biological changes of the cells were observed in which VEGF was up-regulated or down-regulated. RESULTS: VEGF-positive rate was 50%, and VEGF was mainly localized in the cytoplasm and membrane of the tumor cells, while KDR was mainly located in the membrane of vascular endothelial cells in gastric cancer tissues and peri-cancerous tissue. In 2 cases of 50 specimens, the gastric cancer cells expressed KDR, localized in both the cytoplasm and membrane. Introduction of VEGF(165) antisense into human gastric cancer cells (SGC-7901, immunofluorescence intensity, 31.6%)) resulted in a significant reduction in VEGF-specific messenger RNA and total and cell surface VEGF protein (immunofluorescence intensity, 8.9%) (P【0.05). Conversely, stable integration of VEGF(165) in the sense orientation resulted in an increase in cellular and cell surface VEGF (immunofluorescence intensity, 75.4%) (P【0.05). Lowered VEGF levels were associated with a marked decrease in the growth of nude mouse xenografted tumor (at 33 days postimplantation, tumor volume: 345.40 +/- 136.31 mm3)(P【0.05 vs control SGC-7901 group: 1534.40 +/- 362.88 mm3), whereas up-regulation of VEGF resulted in increased xenografted tumor size (at 33 days postimplantation, tumor volume: 2350.50 +/- 637.70 mm3) (P【0.05 vs control SGC-7901 group). CONCLUSION: This study provides direct evidence that VEGF plays an important role in the oncogenesis of human gastric cancer.

Diagnostic value of serum vascular endothelial growth factor and interleukin-17 in primary hepatocellular carcinoma

BACKGROUND Despite significant advancements in the medical treatment of primary hepato-cellular carcinoma(PHC)in recent years,enhancing therapeutic effects and im-proving prognosis remain substantial challenges worldwide.AIM To investigate the expression levels of serum vascular endothelial growth factor(VEGF)and interleukin(IL)-17 in patients with PHC and evaluate their diagnostic value while exploring their relationship with patients’clinical characteristics.METHODS The study included 50 patients with confirmed PHC who visited Wuhan Han-yang Hospital from January 2021 to January 2022,and 50 healthy individuals from the same period served as the control group.Serum VEGF and IL-17 levels in both groups were measured by Enzyme-Linked Immunosorbent Assay,and their diagnostic value was assessed using receiver operating characteristic(ROC)curves.Pearson correlation analysis was performed to examine the relationship between serum VEGF and IL-17 levels.Pathological data of the PHC patients were analyzed to determine the relationship between serum VEGF and IL-17 levels and pathological characteristics.RESULTS Serum VEGF and IL-17 levels were significantly higher in the study group com-pared to the control group(P<0.05).No significant association was observed between serum VEGF and IL-17 levels and gender,age,combined cirrhosis,tumor diameter,or degree of differentiation(P>0.05).However,there was a significant relationship between clinical TNM stage,tumor metastasis,and serum VEGF and IL-17 levels(P<0.05).Correlation analysis revealed a positive correlation between serum VEGF and IL-17(P<0.05).ROC analysis demonstrated that both serum VEGF and IL-17 had good diagnostic efficacy for PHC.CONCLUSION Serum VEGF and IL-17 levels were significantly higher in PHC patients compared to healthy individuals.Their levels were closely related to pathological features such as tumor metastasis and clinical TNM stage,and there was a significant positive correlation between VEGF and IL-17.These biomarkers may serve as valuable reference in-dicators for the early diagnosis and treatment guidance of PHC.

Expression and significance of pigment epithelium-derived factor and vascular endothelial growth factor in colorectal adenoma and cancer

BACKGROUND The incidence and mortality of colorectal cancer(CRC)are among the highest in the world,and its occurrence and development are closely related to tumor neovascularization.When the balance between pigment epithelium-derived factors(PEDF)that inhibit angiogenesis and vascular endothelial growth factors(VEGF)that stimulate angiogenesis is broken,angiogenesis is out of control,resulting in tumor development.Therefore,it is very necessary to find more therapeutic targets for CRC for early intervention and later treatment.AIM To investigate the expression and significance of PEDF,VEGF,and CD31-stained microvessel density values(CD31-MVD)in normal colorectal mucosa,adenoma,and CRC.METHODS In this case-control study,we collected archived wax blocks of specimens from the Digestive Endoscopy Center and the General Surgery Department of Chengdu Second People's Hospital from April 2022 to October 2022.Fifty cases of specimen wax blocks were selected as normal intestinal mucosa confirmed by electronic colonoscopy and concurrent biopsy(normal control group),50 cases of specimen wax blocks were selected as colorectal adenoma confirmed by electronic colonoscopy and pathological biopsy(adenoma group),and 50 cases of specimen wax blocks were selected as CRC confirmed by postoperative pathological biopsy after inpatient operation of general surgery(CRC group).An immunohistochemical staining experiment was carried out to detect PEDF and VEGF expression in three groups of specimens,analyze their differences,study the relationship between the two and clinicopathological factors in CRC group,record CD31-MVD in the three groups,and analyze the correlation of PEDF,VEGF,and CD31-MVD in the colorectal adenoma group and the CRC group.The F test or adjusted F test is used to analyze measurement data statistically.Kruskal-Wallis rank sum test was used between groups for ranked data.The chi-square test,adjusted chi-square test,or Fisher's exact test were used to compare the rates between groups.All differences between groups were compared using the Bonferroni method for multiple comparisons.Spearman correlation analysis was used to test the correlation of the data.The test level(α)was 0.05,and a two-sided P<0.05 was considered statistically significant.RESULTS The positive expression rate and expression intensity of PEDF were gradually decreased in the normal control group,adenoma group,and CRC group(100%vs 78%vs 50%,χ^(2)=34.430,P<0.001;++~++vs+~++vs-~+,H=94.059,P<0.001),while VEGF increased gradually(0%vs 68%vs 96%,χ^(2)=98.35,P<0.001;-vs-~+vs++~+++,H=107.734,P<0.001).In the CRC group,the positive expression rate of PEDF decreased with the increase of differen-tiation degree,invasion depth,lymph node metastasis,distant metastasis,and TNM stage(χ^(2)=20.513,4.160,5.128,6.349,5.128,P<0.05);the high expression rate of VEGF was the opposite(χ^(2)=10.317,13.134,17.643,21.844,17.643,P<0.05).In the colorectal adenoma group,the expression intensity of PEDF correlated negatively with CD31-MVD(r=-0.601,P<0.001),whereas VEGF was not significantly different(r=0.258,P=0.07).In the CRC group,the expression intensity of PEDF correlated negatively with the expression intensity of CD31-MVD and VEGF(r=-0.297,P<0.05;r=-0.548,P<0.05),while VEGF expression intensity was positively related to CD31-MVD(r=0.421,P=0.002).CONCLUSION It is possible that PEDF can be used as a new treatment and prevention target for CRC by upregulating the expression of PEDF while inhibiting the expression of VEGF.

Transforming growth factor-β1 and vascular endothelial growth factor levels in senile acute myeloid leukemia and correlation with prognosis

BACKGROUND Acute myeloid leukemia(AML)is a disease in which immature hematopoietic cells accumulate in the bone marrow and continuously expand,inhibiting hematopoiesis.The treatment and prognosis of this disease have always been unsatisfactory.AIM To investigate the correlation between vascular endothelial growth factor(VEGF)and transforming growth factor-β1(TGFβ1)expression and prognosis in older adults with AML.METHODS This study enrolled 80 patients with AML(AML group),including 36 with complete response(AML-CR),23 with partial response(AML-PR),and 21 with no response(AML-NR).The expression levels of VEGF and TGFβ1 were detected by reverse transcription polymerase chain reaction in bone marrow mononuclear cells isolated from 56 healthy controls.Kaplan-Meier analysis was performed to assess overall survival(OS)and progression-or disease-free survival(DFS).Prognostic risk factors were analyzed using a Cox proportional hazards model.RESULTS The AML group showed a VEGF level of 2.68±0.16.VEGF expression was lower in patients with AML-CR than those with AML-PR or AML-NR(P<0.05).TGFβ1 expression in the AML group was 0.33±0.05.Patients with AML-CR showed a higher TGFβ1 expression than those with AML-PR or AML-NR(P<0.05).VEGF and TGFβ1 expression in patients with AML was significantly correlated with the counts of leukocytes,platelets,hemoglobin,and peripheral blood immature cells(P<0.05);Kaplan-Meier survival analysis revealed that patients with high TGFβ1 expression had better OS and DFS than those with low TGFβ1 expression(P<0.05),whereas patients with low VEGF levels showed better OS and DFS than those with high VEGF levels(P<0.05).VEGF,TGFβ1,and platelet count were identified by the Cox proportional hazards model as independent risk factors for OS(P<0.05),while VEGF,TGFβ1,and white blood cell count were independent risk factors for DFS(P<0.05).CONCLUSION Decreased VEGF expression and increased TGFβ1 expression in patients with AML provide valuable references for determining and individualizing clinical treatment strategies.

Hypoxia-inducible factor 1alpha and vascular endothelial growth factor in Glioblastoma Multiforme:a systematic review going beyond pathologic implications

Glioblastoma multiforme(GBM)is an aggressive primary brain tumor characterized by extensive heterogeneity and vascular proliferation.Hypoxic conditions in the tissue microenvironment are considered a pivotal player leading tumor progression.Specifically,hypoxia is known to activate inducible factors,such as hypoxia-inducible factor 1alpha(HIF-1α),which in turn can stimulate tumor neo-angiogenesis through activation of various downward mediators,such as the vascular endothelial growth factor(VEGF).Here,we aimed to explore the role of HIF-1α/VEGF immunophenotypes alone and in combination with other prognostic markers or clinical and image analysis data,as potential biomarkers of GBM prognosis and treatment efficacy.We performed a systematic review(Medline/Embase,and Pubmed database search was completed by 16th of April 2024 by two independent teams;PRISMA 2020).We evaluated methods of immunoassays,cell viability,or animal or patient survival methods of the retrieved studies to assess unbiased data.We used inclusion criteria,such as the evaluation of GBM prognosis based on HIF-1α/VEGF expression,other biomarkers or clinical and imaging manifestations in GBM related to HIF-1α/VEGF expression,application of immunoassays for protein expression,and evaluation of the effectiveness of GBM therapeutic strategies based on HIF-1α/VEGF expression.We used exclusion criteria,such as data not reporting both HIF-1αand VEGF or prognosis.We included 50 studies investigating in total 1319 GBM human specimens,18 different cell lines or GBM-derived stem cells,and 6 different animal models,to identify the association of HIF-1α/VEGF immunophenotypes,and with other prognostic factors,clinical and macroscopic data in GBM prognosis and therapeutic approaches.We found that increased HIF-1α/VEGF expression in GBM correlates with oncogenic factors,such as miR-210-3p,Oct4,AKT,COX-2,PDGF-C,PLDO3,M2 polarization,or ALK,leading to unfavorable survival.Reduced HIF-1α/VEGF expression correlates with FIH-1,ADNP,or STAT1 upregulation,as well as with clinical manifestations,like epileptogenicity,and a favorable prognosis of GBM.Based on our data,HIF-1αor VEGF immunophenotypes may be a useful tool to clarify MRI-PET imaging data distinguishing between GBM tumor progression and pseudoprogression.Finally,HIF-1α/VEGF immunophenotypes can reflect GBM treatment efficacy,including combined first-line treatment with histone deacetylase inhibitors,thimerosal,or an active metabolite of irinotecan,as well as STAT3 inhibitors alone,and resulting in a favorable tumor prognosis and patient survival.These data were supported by a combination of variable methods used to evaluate HIF-1α/VEGF immunophenotypes.Data limitations may include the use of less sensitive detection methods in some cases.Overall,our data support HIF-1α/VEGF’s role as biomarkers of GBM prognosis and treatment efficacy.

Analyzing the pharmacological substances and targets of Xuefu Zhuyu decoction in hypertensive vascular endothelial cells

Background:Xuefu Zhuyu decoction(XFZY)could significantly improve the function of hypertensive vascular endothelial cells,but the targets and mechanism are not clear.This study is to analyze the pharmacological substances and targets of Xuefu Zhuyu decoction in hypertensive vascular endothelial cells.Methods:This study used Xuefu Zhuyu decoction to intervene human umbilical vein endothelial cells incubated by hypertensive patients’serum,then detected the function of vascular endothelial cells.The aqueous extract of XFZY was analyzed and validated by liquid chromatography-mass spectrometry technology;Finally,macromolecular docking technology was used to analyze the potential active substances and targets of XFZY in the prevention and treatment of hypertension.Results:Compared with the model group,the XFZY group showed a significant increase in NO expression(P<0.01)and a significant decrease in ET-1 expression(P<0.001);and the expression of BIP,P-JNK,CHOP,and BAX in XFZY group cells was significantly decreased(P<0.001),while the expression of JNK and BCL2 was significantly increased(P<0.001).19 main compounds were identified in XFZY and there were 3 pairs of molecular complexes with high affinity for markers of the endoplasmic reticulum stress,including BIP-Hesperidin complex,BIP-HSYA complex and JNK-Naringin complex.Conclusion:This study analyzed the potential pharmacodynamic substance and targets of Xuefu Zhuyu decoction in improving the function of hypertensive vascular endothelial cells,which could provide a scientific basis for the future molecular mechanism of XFZY in treating hypertension.

IMPACTS ON THE EXPRESSION OF VASCULAR ENDOTHELIAL GROWTH FACTOR IN ISCHEMIC BRAIN TISSUE TREATED WITH CLUSTER PUNC-TURE ON SCALP ACUPOINTS IN RATS

Objective To probe into the impacts on the expression of vascular endothelial growth factor （VEGF） in ischemic brain tissue treated with cluster puncture on scalp acupoints in rats, Methods 128 rats were randomized into pseudo-operation group, model group, scalp-needling group and cluster-needling group, In scalp-needling group, penetration method was used on the focal side from Bǎihuì （百会 GV20） to Qǔbīn （曲鬓 GB7）, and in cluster-needling group, penetration method was used on both sides from GV20 to GB7, and a common needling on GV 20. Needles were punctured 2 mm in depth, constantly rotated for 10min, retained for 2 h. Immunohistrochemical method was applied to determine VEGF expression and microvessel density （MVD）. Results With the intervention of cluster puncture on scalp acupoints, VEGF： expressions on every time-spot after ischemia were enhanced apparently, superior to those in scalp-needling group. On the three time-spots of the 7^th, 14^th and 21^st days, MVD was increased after cluster puncture on scale acupoints, superior to those in scalp-needling group. Conclusion Cluster puncture on scalp acupoints up-regulated VEGF expression and promoted regeneration of microvessel.

Reduction of tumorigenicity of SMMC-7721 hepatoma cells by vascular endothelial growth factor antisense gene therapy

AIM: To test the hypothesis to block VEGF expression of SMMC-7721 hepatoma cells may inhibit tumor growth using the rat hepatoma model. METHODS: Amplify the 200 VEGF cDNA fragment and insert it into human U6 gene cassette in the reverse orientation transcribing small antisense RNA which could specifically interact with VEGF165, and VEGF121 mRNA. Construct the retroviral vector containing this antisense VEGF U6 cassette and package the replication-deficient recombinant retrovirus. SMMC-7721 cells were transduced with these virus and positive clones were selected with G418. PCR and Southern blot analysis were performed to determine if U6 cassette integrated into the genomic DNA of positive clone. Transfected tumor cells were evaluated for RNA expression by ribonuclease protection assays. The VEGF protein in the supernatant of parental tumor cells and genetically modified tumor cells was determined with ELISA. In vitro and in vivo growth properties of antisense VEGF cell clone in nude mice were analyzed. RESULTS: Restriction enzyme digestion and PCR sequencing verified that the antisense VEGF RNA retroviral vector was successfully constructed.After G418 selection, resistant SMMC-7721 cell clone was picked up. PCR and Southern blot analysis suggested that U6 cassette was integrated into the cell genomic DNA. Stable SMMC-7721 cell clone transduced with U6 antisense RNA cassette could express 200 bp small antisense VEGF RNA and secrete reduced levels of VEGF in culture condition. Production of VEGF by antisense transgene-expressing cells was 65+/-10 ng/L per 10(6) cells, 42045 ng/L per 10(6) cells in sense group and 485+/-30 ng/L per 10(6) cells in the negative control group, (P【 0.05). The antisense-VEGF cell clone appeared phenotypically indistinguishable from SMMC-7721 cells and SMMC-7721 cells transfected sense VEGF. The growth rate of the antisense-VEGF cell clone was the same as the control cells. When S.C. was implanted into nude mice, growth of antisense-VEGF cell lines was greatly inhibited compared with control cells. CONCLUSION: Expression of antisense VEGF RNA in SMMC-7721 cells could decrease the tumorigenicity, and antisense-VEGF gene therapy may be an adjuvant treatment for hepatoma.

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）

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.

Streptozotocin induced diabetic retinopathy in rat and the expression of vascular endothelial growth factor and its receptor

·AIM: To establishtherat model of streptozotocin (STZ)- induced diabetic retinopathy (DR), which is the most common cause of visual loss and blindness in patients with diabetes, and observe the gene expression of vascular endothelial growth factor (VEGF) and its receptors during the development of DR. ·METHODS: A rat model of diabetes was established by intraperitoneal injection of STZ. The diabetic rats were housed for 2, 3 and 4 months after the development of diabetes. Retinal histopathological observation was performed. The retinal vessels were observed by immunofluorescence staining by CD31. The mRNA expression of VEGF, VEGF receptor 1 and 2 (VEGFR1/2) in rat retina was detected by reverse transcription - polymerase chain reaction (RT-PCR) analysis. · RESULTS: Retinal histopathological observation showed the morphological changes of inner nuclear layer (INL) and outer nuclear layer (ONL) at any time -point, and also demonstrated the increased new vessels at both 3, 4 months after the development of diabetes. The CD31 staining results showed that the number of vessels was increased in the retinas of diabetic rats at both 3 and 4 months after the development of diabetes. As compared to the normal rats, the mRNA expression of VEGF was increased in retinas of diabetic rats at 3 months after the development of diabetes, while VEGFR1 and VEGFR2 mRNA expression was increased at 2, 3 and 4 months after the development of diabetes.·CONCLUSION:Takentogether,ourresultsdemonstrated that DR was occurred at 3 months after the development of diabetes, and the mRNA expression of VEGF, VEGFR1 and VEGFR2 were increased in the process of DR. The present study further evidenced the involvement of VEGF and its receptors in the process of DR. ·

Post-transcriptional regulation of vascular endothelial growth factor: Implications for tumor angiogenesis

Vascular endothelial growth factor （VEGF） is a potent secreted mitogen critical for physiologic and tumor angiogenesis. Regulation of VEGF occurs at several levels, including transcription, mRNA stabilization, translation, and differential cellular localization of various isoforms. Recent advances in our understanding of post-transcriptional regulation of VEGF include identification of the stabilizing mRNA binding protein, HuR, and the discovery of internal ribosomal entry sites in the 5＇UTR of the VEGF mRNA. Monoclonal anti-VEGF antibody was recently approved for use in humans, but suffers from the need for high systemic doses. RNA interference （RNAi） technology is being used in vitro and in animal models with promising results. Here, we review the literature on post-transcriptional regulation of VEGF and describe recent progress in targeting these mechanisms for therapeutic benefit.

Expression of vascular endothelial growth factor and its receptors VEGFR-1 and 2 in gastrointestinal stromal tumors,leiomyomas and schwannomas

AIM： To investigate the role of vascular endothelial growth factor （VEGF/and its receptors VEGFR-1 and 2 in the growth and differentiation of gastrointestinal strornal tumors （GISTs）. METHODS： Thirty-three GISTs, 15 leiomyomas and 6 schwannomas were examined by immunohistochemistry in this study. RESULTS： VEGF protein was expressed in the cytoplasm of tumor cells, and VEGFRol and 2 were expressed both in the cytoplasm and on the membrane of all tumors. Irnrnunohistochernical staining revealed that 26 GISTs （78.8%）, 9 leiornyornas （60.0%） and 3 schwannornas （50.0%/were positive for VEGF; 24 GISTs （72.7%/, 12 leiornyornas （80.0%） and 4 schwannornas （66.7%） were positive for VEGFR-1; 30 GISTs （90.9%/, 5 leiornyornas （33.3%/and 4 schwannornas （66.7%） were positive for VEGFR-2. VEGFR-2 expression was statistically different between GISTs and leiomyomas （P 〈 0.0001）. However, there was no correlation between the expression of VEGF pathway componenets and the clinical risk categories. CONCLUSION： Our results suggest that the VEGF pathway may play an important role in the differentiation of GISTs, leiomyomas and schwannomas.

Effects of endostatin on expression of vascular endothelial growth factor and its receptors and neovascularization in colonic carcinoma implanted in nude mice

AIM:To investigate the antiangiogenic effects of endostatin on colonic carcinoma cell line implanted in nude mice and its mechanism. METHODS:Nude mice underwent subcutaneous injection with LS-174t colonic carcinoma cell line to generate carcinoma and were randomly separated into two groups.Mice received injection of vehicle or endostatin every day for two weeks. After the tumor was harvested,the tumor volumes were determined,and the expressions of CD34,VEGF and FIk-1 were examined by immunohistochemical method. RESULTS:Tumor volume was significantly inhibited in the endostatin group(84.17%)and tumor weight was significantly inhibited in the endostatin group(0.197±0.049) compared to the control group(1.198±0.105)(F=22.56, P=0.001),microvessel density(MVD)was significantly decreased in the treated group(31.857±3.515)compared to the control group(100.143±4.290)(F=151.62,P<0.001). Furthermore,the expression of FIk-1 was significantly inhibited in the treated group(34.29%) ompared to the control group(8.57%)(X^2=13.745,P=0.001).However no significant decrease was observed in the expression of vascular endothelial growth factor(VEGF)between these two groups(X^2=0.119,P=0.730). CONCLUSION:Endostatin can inhibit tumor growth and angiogenesis by blocking Vegf/FIk-1 pathway.This experiment provides the theory basis for developing a new anti-carcinoma drug through studying the properties of anti-angiogenesis inhibitors.

Construction of human eukaryotic expression plasmid vascular endothelial growth factor 165 and its expression in transfected vascular smooth muscles

BACKGROUND: The highly specific vascular endothelialgrowth factor (VEGF) induces the growth of vascular en-dothelial cell. This study was to construct the eukaryoticexpression plasmid of vascular endothelial growth factorl65(VEGF165) and observe its expression in vascular smoothmuscles (VSMCs).METHODS: The primers were designed and synthesizedaccording to the gene sequences of human VEGF165. TheVEGF165 gene was obtained from umbilic artery tissue bythe method of RT-PCR, then it was cloned to eukaryoticexpression plasmid pBudCE4.1 by recombination strategy.The eukaryotic expression plasmid named pBudCE4.1/VEGF165 was identified by restriction enzyme digestion,and was sequenced. The pBudCE4.1/VEGF165 was trans-fected into VSMCs by using lipofection. The VEGF165 ex-pression of mRNA and protein was detected by RT-PCRand Western blot respectively.RESULTS: VEGF165 was shown about 576bp by RT-PCR.Sequencing revealed the amplified VEGF165 gene was iden-tical with that in the GeneBank. Restrictive enzyme (HindBam HI) digestion analysis showed that recombinantexpression plasmid pBudCE4. l/tVEGF165 had been con-structed successfully. The expression of VEGF165 at mRNAand protein levels in the transformed VSMCs had beendemonstrated by RT-PCR and Western blot.CONCLUSIONS: The recombinant eukaryotic expressionplasmid pBudCE4.1/VEGF165 has been successfully con-structed and expressed in transformed VSMCs. The presentstudy has laid a foundation for VEGF165 gene therapy ofvascular stenosis in the transplant organ.

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.

Elevation of vascular endothelial growth factor production and its effect on revascularization and function of graft islets in diabetic rats

AIM: To determine whether the elevated vascular endothelial growth factor (VEGF) expression produced by the transfected vascular endothelial cells (VECs) could stimulate angiogenesis of the graft islets and exert its effect on the graft function. METHODS: Thirty diabetic recipient rats were divided into three groups (n = 10 per group). In the control group,300 IEQ islets were transplanted in each rat under the capsule of the right kidney,which were considered as marginal grafts. In the VEC group,VEC together with the islets were transplanted in each rat. In the VEGF group,VEC transfected by pIRES2-EGFP/ VEGF165 plasmid and the islets were transplanted in each rat. Blood glucose and insulin levels were evaluated every other day after operation. Intravenous glucose tolerance test (IVGTT) was performed 10 d after the transplantation. Hematoxylin and eosin (HE) staining was used to evaluate the histological features of the graft islets. Immunohistochemical staining was used to detect insulin-6,VEGF and CD34 (MVD) expression in the graft islets. RESULTS: Blood glucose and insulin levels in the VEGF group restored to normal 3 d after transplantation. In contrast,diabetic rats receiving the same islets with or without normal VECs displayed moderate hyperglycemia and insulin,without a significant difference between these two groups. IVGTT showed that both the amplitude of blood glucose induction and the kinetics of blood glucose in the VEGF group restored to normal after transplantation. H&E and immunohistochemical staining showed the presence of a large amount of graft islets under the capsule of the kidney,which were positively stained with insulin-6 and VEGF antibodies in the VEGF group. In the cell masses,CD34-stained VECs were observed. The similar masses were also seen in the other two groups,but with a fewer positive cells stained with insulin-6 and CD34 antibodies. No VEGF-positive cells appeared in these groups. Microvessel density (MVD) was significantly higher in the VEGF group compared to the other two groups. CONCLUSION: Elevated VEGF production by trans-fected vascular endothelial cells in the site of islet transplantation stimulates angiogenesis of the islet grafts. The accelerated islet revascularization in early stage could improve the outcome of islet transplantation,and enhance the graft survival.

Effect of c-fos antisense probe on prostaglandin E_2-induced upregulation of vascular endothelial growth factor mRNA in human liver cancer cells

AIM: To examine the effect of prostaglandin E2 (PGE2) on the expression of vascular endothelial growth factor (VEGF) mRNA in the human hepatocellular carcinoma (HCC) HepG2 cells and the possible involvement of c-fos protein in this process.METHODS: Human HCC HepG2 cells were divided into three groups treated respectively with PGE2, a combination of PGE2 and c-fos antisense oligodeoxynucleotide (ASO),and PGE2 plus c-fos sense oligodeoxynudeotide (SO). The expression of VEGF mRNA in HepG2 cells after different treatments was detected by reverse transcriptase-polymerase chain reaction (RT-PCR). The relative expression level of VEGF mRNA in HepG2 cells in each group was measured.RESULTS: Administration of PGE2 resulted in an increased expression of c-fosand VEGF mRNA in HepG2 cells. The relative expression level of c-fos mRNA reached the peak at 3 h (68.4±4.7%) after PGE2 treatment, which was significantly higher than that at 0 h (20.6±1.7%, P＜0.01).Whereas, the highest expression level of VEGF mRNA was observed at 6 h (100.5±6.1%) after PGE2 treatment, which was significantly higher than that at 0 h (33.2±2.4%,P＜0.01). C-fos ASO significantly reduced PGE2-induced VEGF mRNA expression in HepG2 cells.CONCLUSION: PGE2 increases the expression and secretion of VEGF in HCC cells by activating the transcription factor c-fos, promotes the angiogenesis of HCC and plays an important role in the pathogenesis of liver cancer.

Influence of vascular endothelial growth factor and radiation on gap junctional intercellular communication in glioblastoma multiforme cell lines

Glioblastoma multiforme （GBM） is a highly aggressive glial brain tumor with an unfavorable prognosis despite all current therapies including surgery, radiation and chemotherapy. One characteristic of this tumor is a strong synthesis of vascular endothelial growth factor （VEGF）, an angiogenesis factor, followed by pronounced vascularization. VEGF became a target in the treatment of GBM, for example with bevacizumab or the tyrosine kinase inhibitor axitinib, which blocks VEGF receptors. To improve patients＇ prognosis, new targets in the treatment of GBM are under investigations. The role of gap junctions in GBM remains un- known, but some experimental therapies affect these intercellular channels to treat the tumor. Gap junctions are composed of connexins to allow the transport of small molecules between adjacent cells through gap junc- tional intercellular communication （GJIC）. Based on data derived from astrocytes in former studies, which show that VEGF is able to enhance GJIC, the current study analyzed the effects of VEGF, radiation therapy and VEGF receptor blockade by axitinib on GJIC in human GBM cell lines U-87 and U-251. While VEGF is able to induce GJIC in U-251 cells but not in U-87 cells, radiation enhances GJIC in both cell lines. VEGF reocptor blockade by axitinib diminishes radiation induced effects in U-251 partially, while increases GJIC in U-87 cells. Our data indicate that VEGF and radiation are both modifying components of GJ1C in pathologic brain tumor tissue.