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.

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.

Vascular endothelial growth factor/connective tissue growth factor and proteomic analysis of aqueous humor after intravitreal conbercept for proliferative diabetes retinopathy

AIM:To investigate the role of connective tissue growth factor(CTGF)and vascular endothelial growth factor(VEGF)in the protein profile of the aqueous humor in patients with proliferative diabetic retinopathy(PDR)following intravitreal injection of conbercept.METHODS:This study included 72 PDR patients and 8 cataract patients as controls.PDR patients were divided into 3 groups according to the intervals of 3,5,and 7d between intravitreal conbercept(IVC,0.5 mg/0.05 mL)injection and pars plana vitrectomy(PPV)performed.Aqueous humor samples were collected before and after IVC and PPV for VEGF and CTGF levels detected with enzyme-linked immunosorbent assay(ELISA).The differential proteomics of 10 patients who underwent PPV surgery 5d after IVC and 8 normal controls was studied,Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)analysis were performed on the data,and the protein interaction network of 23 differential proteins was studied.RESULTS:Post-IVC,VEGF levels decreased and CTGF levels increased significantly in aqueous humor,with the CTGF/VEGF ratio rising significantly at all intervals.Liquid chromatography tandem mass spectrometry(LC-MS/MS)identified differentially expressed proteins between preand post-IVC samples.GO and KEGG analyses revealed involvement in immune response,stress response,complement and coagulation cascades,ferroptosis,and PPAR signaling pathways.PPI analysis highlighted key proteins like APOA1,C3,and transferrin(TF).ELISA assay confirmed the differential expression of proteins such as HBA1,SERPINA1,COL1A1,and ACTB,with significant changes in the IVC groups.CONCLUSION:The study demonstrates that IVC effectively reduces VEGF levels while increasing CTGF levels,thereby modifying the CTGF/VEGF ratio,and IVC significantly alters the protein profile in the aqueous humor of patients with PDR.Proteomic analysis reveals that these changes are associated with critical biological pathways and protein interactions involved in immune response,stress response,and cellular metabolism.

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.

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.

Anti-vascular endothelial growth factor drugs combined with laser photocoagulation maintain retinal ganglion cell integrity in patients with diabetic macular edema: study protocol for a prospective, non-randomized, controlled clinical trial

The integrity of retinal ganglion cells is tightly associated with diabetic macular degeneration that leads to damage and death of retinal ganglion cells,affecting vision.The major clinical treatments for diabetic macular edema are anti-vascular endothelial growth factor drugs and laser photocoagulation.However,although the macular thickness can be normalized with each of these two therapies used alone,the vision does not improve in many patients.This might result from the incomplete recovery of retinal ganglion cell injury.Therefore,a prospective,non-randomized,controlled clinical trial was designed to investigate the effect of anti-vascular endothelial growth factor drugs combined with laser photocoagulation on the integrity of retinal ganglion cells in patients with diabetic macular edema and its relationship with vision recovery.In this trial,150 patients with diabetic macular edema will be equally divided into three groups according to therapeutic methods,followed by treatment with anti-vascular endothelial growth factor drugs,laser photocoagulation therapy,and their combination.All patients will be followed up for 12 months.The primary outcome measure is retinal ganglion cell-inner plexiform layer thickness at 12 months after treatment.The secondary outcome measures include retinal ganglion cell-inner plexiform layer thickness before and 1,3,6,and 9 months after treatment,retinal nerve fiber layer thickness,best-corrected visual acuity,macular area thickness,and choroidal thickness before and 1,3,6,9,and 12 months after treatment.Safety measure is the incidence of adverse events at 1,3,6,9,and 12 months after treatment.The study protocol hopes to validate the better efficacy and safety of the combined treatment in patients with diabetic macula compared with the other two monotherapies alone during the 12-month follow-up period.The trial is designed to focus on clarifying the time-effect relationship between imaging measures related to the integrity of retinal ganglion cells and best-corrected visual acuity.The trial protocol was approved by the Medical Ethics Committee of the Affiliated Hospital of Beihua University with approval No.(2023)(26)on April 25,2023,and was registered with the Chinese Clinical Trial Registry(registration number:ChiCTR2300072478,June 14,2023,protocol version:2.0).

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.

Efficacy of recombinant human epidermal growth factor plus sodium hyaluronate eye drops in diabetic dry eye post-cataract surgery

BACKGROUND Dry eye syndrome(DES)after diabetic cataract surgery can seriously affect the patient’s quality of life.Therefore,effective alleviation of symptoms in patients with this disease has important clinical significance.AIM To explore the clinical effect of recombinant human epidermal growth factor(rhEGF)plus sodium hyaluronate(SH)eye drops on DES after cataract surgery in patients with diabetes.METHODS We retrospectively evaluated 82 patients with diabetes who experienced DES after cataract surgery at Tianjin Beichen Hospital,Affiliated Hospital of Nankai University between April 2021 and April 2023.They were classified into an observation group(42 cases,rhEGF+SH eye drops)and a control group(40 cases,SH eye drops alone),depending on the different treatment schemes.The therapeutic efficacy,dry eye symptom score,tear film breakup time(TFBUT),basic tear secretion score[assessed using Schirmer I test(SIt)],corneal fluorescein staining(FL)score,tear inflammatory markers,adverse reactions during treat-ment,and treatment satisfaction were compared between the two groups.RESULTS Therapeutic efficacy was higher in the observation group compared with the control group.Both groups showed improved TFBUT and dry eye,as well as improved SIt and FL scores after treatment,with a more pronounced improvement in the observation group.Although no marked differences in adverse reactions were observed between the two groups,treatment satisfaction was higher in the observation group.CONCLUSION rhEGF+SH eye drops rendered clinical benefits to patients by effectively ameliorating dry eye and visual impairment with favorable efficacy,fewer adverse reactions,and high safety levels.Thus,this treatment should be promoted in clinical practice.

Nomogram for predicting short-term response to anti-vascular endothelial growth factor treatment in neovascular age-related macular degeneration:An observational study

BACKGROUND Anti-vascular endothelial growth factor(anti-VEGF)therapy is critical for managing neovascular age-related macular degeneration(nAMD),but understanding factors influencing treatment efficacy is essential for optimizing patient outcomes.AIM To identify the risk factors affecting anti-VEGF treatment efficacy in nAMD and develop a predictive model for short-term response.METHODS In this study,65 eyes of exudative AMD patients after anti-VEGF treatment for≥1 mo were observed using optical coherence tomography angiography.Patients were classified into non-responders(n=22)and responders(n=43).Logistic regression was used to determine independent risk factors for treatment response.A predictive model was created using the Akaike Information Criterion,and its performance was assessed with the area under the receiver operating characteristic curve,calibration curves,and decision curve analysis(DCA)with 500 bootstrap re-samples.RESULTS Multivariable logistic regression analysis identified the number of junction voxels[odds ratio=0.997,95%confidence interval(CI):0.993-0.999,P=0.010]as an independent predictor of positive anti-VEGF treatment outcomes.The predictive model incorporating the fractal dimension,number of junction voxels,and longest shortest path,achieved an area under the curve of 0.753(95%CI:0.622-0.873).Calibration curves confirmed a high agreement between predicted and actual outcomes,and DCA validated the model's clinical utility.CONCLUSION The predictive model effectively forecasts 1-mo therapeutic outcomes for nAMD patients undergoing anti-VEGF therapy,enhancing personalized treatment planning.

Advances of Study on Recombinant Vascular Endothelial Growth Factor

血管内皮生长因子 (Vascularendothelialgrowthfactor,VEGF)是 1 989年Ferrara等在牛垂体滤泡星状细胞体外培养液中首先纯化出来的。到目前为止已经发现了六个VEGF异构体 ,包括VEGF1 2 1 ,VEGF1 45,VEGF1 6 5,VEGF1 83 ,VEGF1 89,VEGF2 0 6。已经发现高亲和力的VEGF受体有两类 ,分别为Flt 1 (Fms liketyrosinekinase)和KDR(kinasedomainregion)。重点介绍了VEGF两个方面的应用前景。一方面用于治疗下肢缺血和心脏病 。

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.

Quantitative analysis of vascular endothelial growth factor, microvascular density and their clinicopathologic features in human hepatocellular carcinoma

BACKGROUND: Angiogenesis is known to be essential to the survival, growth, invasion, and metastasis of tumor cells. Vascular endothelial growth factor (VEGF) are an important angiogenic factor regulating tumor angiogenesis, but its significance and tumor pathologic features are un- clear in hepatocellular carcinoma (HCC). In the present study, we analyzed expression of tissue VEGF, alteration of microvascular density (MVD) in microvessel angiogenesis, development and metastasis of HCC, and level of serum VEGF in differential diagnosis of benign and malignant liv- er diseases. METHODS: Tumor specimens were prospectively collected from HCC patients undergoing resection. Total RNAs were extracted and the expression levels were detected from different parts of HCC tissues. The cellular distributions of VEGF and MVD of liver tumors and their paracancerous and distal cancerous tissues were investigated by streptavi- din peroxidase (S-P) immunohistochemistry, respectively. The VEGF levels of circulating blood and hepatoma tissues were measured by enzyme-linked immunosorbent assay. RESULTS: The incidence of VEGF expression was 63.9% in HCCs (23/36 cases), 78.3% in non-encapsulated HCCs (18/23), and 90.9% in HCCs with extrahepatic metastasis (10/11), respectively. The VEGF expression was tightly correlated with MVD (P <0.01). The MVD in HCC with metastasis, low differentiation or non-encapsulation was significantly higher than that in HCC with intact capsule, high differentiation, or no metastasis. No significant diffe- rence was found between VEGF, MVD, tumor size, and hepatitis virus infection. The level of total RNA in HCC tis- sues was significantly lower but the VEGF level significantly higher than those in paracancerous or distal cancerous ones (P<0.01). The abnormal expression levels of VEGF in sera of HCC patients were directly correlated with the me- tastasis and recurrence of tumors. CONCLUSION: The high expression of VEGF and abnor- mality of tissue MVD are useful predictors for vascular inva- sion and metastasis of liver tumors.

Quantitative analysis using ELISA of vascular endothelial growth factor and basic fibroblast growth factor in human colorectal cancer,liver metastasis of colorectal cancer and hepatocellular carcinoma

Angiogenesis consists of the sprouting of capillaries from pre-existing vessels. It is well-known that tumor growth is angiogenesis-dependent. Vascular endothelial growth factor （VEGF） and basic fibroblast growth factor （bFGF） stimulated vascular endothelial cell proliferation and are involved in the neoplastic angiogenesis of several types of tumors including those of the intestinal tract. Authors usually investigated VEGF and using immunohistochemistry bFGF protein expressions or Western blotting and VEGF and bFGF transcripts using reverse transcriptase Dolymerase chain reaction （RT-PCR）.

Expression of Livin and vascular endothelial growth factor in different clinical stages of human esophageal carcinoma

AIM： To investigate the role of Livin and vascular endothelial growth factor （VEGF） in human esophageal carcinoma, and analyze its relationship to clinical stages.METHODS： Expression of Livin in fresh esophageal cancer tissues was detected by immunohistochemistry （IHC）, Western blotting and reverse transcriptasepolyrnerase chain reaction （RT-PCR）, and VEGF by Western blotting and RT-PCR. All statistical analyses were performed by SPSS version 11.0. RESULTS： Livin positivity was also significantly correlated with tumor stages, increasing with tumor progression. Expression of Livin and VEGF increased with the process of esophageal carcinoma. In the fourth clinical stage, expression of Livin and VEGF was the most significant. Expression of Livin was positively correlated with VEGF. CONCLUSION： Over-expression of Livin and VEGF contributes to the pathogenesis of esophageal carcinoma.

Activation of STAT3 signaling in human stomach adenocarcinoma drug-resistant cell line and its relationship with expression of vascular endothelial growth factor

AIM: To investigate the difference in activation of STAT3 signaling between two human stomach adenocarcinoma cell lines: 5-fluorouracil resistant cell line and its parental cell line, and to evaluate its relationship with the expression of vascular endothelial growth factor (VEGF). METHODS: Western blot and electrophoretic mobility shift assay (EMSA) were used to detect the expression of phospho-STAT3 protein and constitutive activation of STAT3 in two human stomach adenocarcinoma cell lines, 5-fluorouracil resistant cell line SGC7901/R and its parental cell line SGC7901, respectively. The mRNA expression of VEGF was analysed by semi-quantitative RT-PCR. The expressive intensity of VEGF protein was measured by immunocytochemistry. RESULTS: The expressions of phospho-STATS protein and constitutive activation of STAT3 between two human stomach adenocarcinoma cell lines were different. Compared with the parental cell line SGC7901, the STAT3DNA binding activity and the expressive intensity of phospho-STAT3 protein were lower in the drug-resistant cell line SGC7901/R. The expression levels of VEGF mRNA and its encoded protein were also decreased in drugresistant cell line. CONCLUSION: Over-expression of VEGF may be correlated with elevated STAT3 activation in parental cell line. Lower VEGF expression may be correlated with decreased STAT3 activation in resistant cell line, which may have resulted from negative feedback regulation of STAT signaling.

Expression of thymidine kinase mediated by a novel non-viral delivery system under the control of vascular endothelial growth factor receptor 2 promoter selectively kills human umbilical vein endothelial cells

AIM: To investigate the killing efficiency of a recombinant plasmid containing a thymidine kinase (TK) domain insert driven by the vascular endothelial growth factor receptor 2 (VEGFR2) promoter (KDR) on vascular endothelial cells.METHODS: The KDR-TK fragment was extracted from pBluescript Ⅱ KDR-TK plasmid by enzymatic digestion with Xho I and Sal I. The enhanced green fluorescence protein (EGFP) carrier was extracted from pEGFP by the same procedure. The KDR-TK was inserted into the pEGFP carrier to construct pEGFP-KDR-TK. Using ultrasound irradiation and microbubble, pEGFP-KDR-TK was transferred into human umbilical vein endothelial cells (HUVECs). The transient infection rate was estimated by green fluorescent protein (GFP) expression. Transfected HUVECs, non-transfected HUVECs, and HepG2 cells were cultured in the presence of different concentrations of ganciclovir (GCV), and the killing efficacy of HSV-TK/GCV was analyzed by 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyl tetrazolium bromide (MTT) assay. RESULTS: The recombinant pEGFP-KDR-TK was successfully constructed by inserting the KDR-TK fragment into the pEGFP carrier. Transfected HUVECs showed cytoplasmic green fluorescence, and the transient transfection rate was about 20.3%. Pools of G418-resistant cells exhibited a higher sensitivity to theprodrug/GCV compared to non-transfected HUVECs or non-transfected HepG2 cells, respectively. CONCLUSION: KDR promoter and the suicide gene/prodrug system mediated by diagnostic ultrasound combined with microbubble can significantly kill HUVECs. Such therapy may present a novel and attractive approach to target gene therapy on tumor vessels.

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.

Effect of non-anticoagulant N-desulfated heparin on expression of vascular endothelial growth factor, angiogenesis and metastasis of orthotopic implantation of human gastric carcinoma

AIM： To investigate the effect of N-desulfated heparin on tumor metastasis and angiogenesis, and expression of vascular endothelial growth factor （VEGF） of orthotopic implantation of human gastric carcinoma in male severe combined immune deficiency （SCID） mice. METHODS： Human gastric cancer SGC-7901 cells were orthotopically implanted into the stomach of SC/D mice. The mice were randomly divided into normal saline group and N-desulfated heparin group. One week after operation, the mice in N-desulfated heparin group reo ceived i.v. injections of N-desulfated heparin （Shanghai Institute of Cell Biology, Chinese Academy of Sciences, 10 mg/kg.d） twice weekly for 3 wk. The mice in normal saline group received i.v. injections of normal saline （100 μL） twice weekly for 3 wk. The mice were sacrificed six weeks after implantation. Tumor metastasis was evaluo ated histologically for metastasis under microscope. Intratumoral microvessel density （MVD） and VEGF expression were evaluated immuohistochemically. VEGF mRNA expression in gastric tissue of SC/D mice was detected by real time PCR. RESULTS： The tumor metastasis rate was 80% in normal saline group and 20% in N-desulfated heparin group （P 〈 0.05）. MVD was 8.0 ± 3.1 in normal saline group and 4.3 ± 1.8 in N-desulfated heparin group （P 〈 0.05）. VEGF positive immunostaining was found in cytoplasm of cancer cells. The rate of VEGF positive expression was higher in normal saline group than in N-desulfated hepa- rin treated group （90% vs 20%, P 〈 0.05）. VEGF mRNA expression was significantly inhibited by N-desulfated heparin and was higher in normal saline group than in N-desulfated heparin group （Ct value 19.51 ± 1.01 vs 22.55± 1.36, P 〈 0.05）. N-desulfated heparin significantly inhibited the expression of VEGF mRNA in cancer cells. No bleeding occurred in N-desulfated heparin group. CONCLUSION： N-desulfated heparin can inhibit metastasis of gastric cancer by suppressing tumor VEGF expression and tumor angiogenesis, but has no obvious anticoagulant activity.

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.

Recombinant expression and purification of functional vascular endothelial growth factor-121 in the baculovirus expression system

Objective: To express human Vascular endothelial growth factor121(VEGF121) in insect cells. Methods: A gene construct containing VEGF was cloned in the p Fast Bac-HTA vector, followed by transformation in DH10 BAC. The recombinant bacmid was then extracted, and transfected into Sf9 insect cells. The transfected cells were harvested, and then VEGF expression was confirmed by Western blotting using specific antibodies. The tube formation assay was used for functional assessment of VEGF. Results: Our results showed that VEGF could be successfully expressed in the baculovirus system. Purified VEGF was able to stimulate in vitro tube formation of human endothelial cells. Conclusions: Results from this study demonstrated that the recombinantly-produced VEGF can be considered as a promising candidate for therapeutic purposes.