Effects of Vascular Endothelial Cell Growth Factor on Fibrovascular Ingrowth into Rabbit's Hydroxyapatite Orbital Implant

Summary: The effects of different concentrations of vascular endothelial cell growth factor (VEGF) on the fibrovascular ingrowth into rabbits hydroxyapatite orbital implant were investigated. Twelve New Zealand white rabbits were divided into 3 groups and received hydroxyapatite orbital implant surgery in their right eyes. Before and after the operation, the implants were treated with 10 ng/ml VEGF, 100 ng/ml VEGF, or normal saline as control group. The animals received technetium bones scan at 2, 4, and 6 weeks postoperatively. The mean radioactivity counts within region of interest (ROI) of the surgery eye (R) and the non-surgery eye (L) in the same animal were tested, and the R/L ratios were calculated. The implants were harvested at 6th weeks and examined histopathologically. The results showed that at second week, there was no significant difference in mean R/L ratios between VEGF group and control group (F=2.83, P=0.111); At 4th week (F=7.728, P=0.011) and 6th week (F=7.831, P=0.011) postoperatively, the mean ratios in VEGF groups were significantly higher than that in control group. At 6th week postoperatively, the fibrovascularization rates in VEGF groups were higher than in control group significantly (F=8.711, P=0.008). It was suggested that VEGF could promote the fibrovascular ingrowth into hydroxyapatite orbital implant, thus might shorten the time required for complete vascularization of the HA orbital implant.

Effects of bone marrow-derived mesenchymal stemcells engraftment on vascular endothelial cell growthfactor in lung tissue and plasma at early stage of smoke inhalation injury

BACKGROUND： This study was undertaken to determine the effect of mesenchymal stem cells （MSCs） engraftment on vascular endothelial cell growth factor （VEGF） in lung tissue, plasma and extravascular lung water at early stage of smoke inhalation injury.METHODS： A rabbit smoke inhalation injury model was established using a home-made smoke inhalation injury generator, and rabbits were divided into two groups randomly： a control group （S group, n=32） and a MSCs treatment group （M group, n=32）. 10 ml PBS was injected via the ear marginal vein immediately at injury into the S group. Third generation MSCs with a concentration of 1×107/10 ml PBS were injected via the ear marginal vein immediately at injury into the M group. VEGF in peripheral blood and lung tissue were measured at 0 （baseline）, 2, 4 and 6 hours after injection respectively and analyzed. The right lungs of rabbits were taken to measure lung water mass fraction.RESULTS： In the lung tissue, VEGF decreased gradually in the S group （P〈0.05） and signi？ cantly decreased in the M group （P〈0.05）, but it increased more signi？ cantly than the values at the corresponding time points （P〈0.05）. In peripheral blood, VEGF increased gradually in the S group （P〈0.05） and markedly increased in the M group （P〈0.05）, but it decreased more signi？ cantly than the values at corresponding time points （P〈0.05）.CONCLUSION： MSCs engraftment to smoke inhalation injury could increase VEGF in lung tissue, decrease VEGF in plasma and reduce extravascular lung water, indicating its protective effect on smoke inhalation injury.

Vascular Endothelial Growth Factorl65-regulated Nasopharyngeal Carcinoma Cell Lines Invasion and Migration Involve Expression and Activation of Matrix Metalloproteinase-2

The effect of vascular endothelial growth factor （VEGF） overexpression on matrix metalloproteinase-2 （MMP-2） in nasopharyngeal carcinoma （NPC） cells in vitro and the possible mechanism involved were investigated, and the correlation between the expression of VEGF and MMP-2 in NPC evaluated. The NPC cells were transfected with PAd-trackVEGF165 plasmid. The expression levels of VEGF and MMP-2 mRNA and protein in NPC cells were detected by semi-quantitative RT-PCR and Western blot respectively. It was found that the expression of VEGF and MMP-2 mRNA and protein was significantly increased in NPC cells after transfection of VEGF 165. It was concluded that the expression of VEGF was correlated to the in vitro invasion of NPC cells, and the induction of MMP-2 by VEGF was a key process of NPC cell invasion.

Effect of Fuzhenghuayu decoction on vascular endothelial growth factor secretion in hepatic stellate cells

Objective: To investigate the effect of Fuzhenghuayu decoction on autocrine activation of hepatic stellate cell (HSC). Methods: The drug serum containing Fuzhenghuayu decoction was collected from normal rats, and cul- tured with activated HSC in vitro. The conditioned medium from the drug serum treated HSC was added to primary cultured quiescent HSC. Cell prolifera- tion was assayed by tetrazolium colorimetric test, and the contents of type Ⅰ collagen and vascular endo- thelial growth factor (VEGF) in the supernatant were measured with ELISA. Results: The conditioned medium from activated HSC could stimulate the quiescent HSC proliferation and type Ⅰ collagen secretion. The drug serum inhibi- ted this stimulating action and VEGF secretion from the activated HSC. Conclusion: Fuzhenghuayu decoction acts effectively against the autocrine activation pathway of HSC. The mechanism may be associated with the inhibition of the secretion of VEGF by activated HSC.

Vascular endothelial growth factor 165b expression in stromal cells and colorectal cancer

AIM:To characterize the implications of vascular endothelial growth factor(VEGF)-A in stromal cells and colorectal cancer and the expression of VEGF-A splice variants.METHODS:VEGF-A expression in tumor and stromal cells from 165 consecutive patients with colorectal cancer was examined by immunohistochemistry.The association between VEGF-A expression status and clinicopathological factors was investigated.Twenty freshfrozen samples were obtained for laser capture microdissection to analyze the splice variants of VEGF-A.RESULTS:VEGF-A was expressed in 53.9% and 42.4% of tumor and stromal cells,respectively.VEGF-A expression in tumor cells(t-VEGF-A) was associated with advanced clinical stage(stage 0,1/9;stage 1,2/16;stage 2,32/55;stage 3,38/66;stage 4,16/19,P < 0.0001).VEGF-A expression in stromal cells(s-VEGF-A) increased in the earlier clinical stage(stage 0,7/9;stage 1,6/16;stage 2,33/55;stage 3,22/66;stage 4,5/19;P = 0.004).Multivariate analyses for risk factors of recurrence showed that only s-VEGF-A expression was an independent risk factor for recurrence(relative risk 0.309,95% confidence interval 0.141-0.676,P = 0.0033).The five-year disease-free survival(DFS) rates of t-VEGF-A-positive and-negative cases were 51.4% and 62.9%,respectively.There was no significant difference in t-VEGF-A expression status.The five-year DFS rates of s-VEGF-A-positive and-negative cases were 73.8% and 39.9%,respectively.s-VEGFA-positive cases had significantly better survival than s-VEGF-A-negative cases(P = 0.0005).Splice variant analysis revealed that t-VEGF-A was mainly composed of VEGF165 and that s-VEGF-A included both VEGF165 and VEGF165b.In cases with no venous invasion(v0),the level of VEGF165b mRNA was significantly higher(v0 204.5 ± 122.7,v1 32.5 ± 36.7,v2 2.1 ± 1.7,P = 0.03).The microvessel density tended to be lower in cases with higher VEGF165b mRNA levels.CONCLUSION:s-VEGF-A appears be a good prognostic factor for colorectal cancer and includes VEGF165 and VEGF165b.

Resveratrol Inhibits the Secretion of Vascular Endothelial Growth Factor and Subsequent Proliferation in Human Leukemia U937 Cells

This study examined the effect of resveratrol on the secretion of vascular endothelial growth factor （VEGF） and subsequent proliferation of human leukemia U937 cells, and explored the mechanisms involved. Human leukemia U937 cells were treated with resveratrol of different concentrations （12.5-200 μmol/L） for different time lengths （12-48 h）. The proliferation of the U937 leukemic cells was determined by MTT assay. Apoptosis was observed by Annexin- V-FIFC/PI double staining and flow cytometry （FCM）. Cells cycle was analyzed by PI staining and FCM. The content of VEGF was determined by ELISA. Human umbibical vein endothelial cells were examined for vasoformation in vitro after exposures to resveratrol of various concetrations. The results showed that resveratrol inhibited the proliferation of U937 leukemia cells in a dose- and time-dependent manner. Resveratrol induced apoptosis and S-phase cell cycle arrest in human leukemic U937 cells. Resveratrol inhibited the secretion of VEGF in U937 cells. Resveratrol inhibited the vasoformation of human vein endothelial cells in a dose-dependent manner. It was concluded that resveratrol could down-regulate the secretion of VEGE induce apoptosis and suppress the proliferation of U937 cells.

Effects of Meloxicam on Vascular Endothelial Growth Factor and Angiopoietin-2 Expression in Colon Carcinoma Cell Line HT-29

To investigate the effect of meloxicam, a selected NSAIDs, on cell growth, expression of VEGF and angiopointin-2 （Ang-2） protein in HT-29 cell line, cultured HT-29 cells were treated with meloxicam of various concentrations for various lengths of time. The proliferation of HT-29 was detected by cell counting kit-8 （CCK-8）, the cell cycle was determined by flow cytometer and the levels of VEGF and Ang-2 protein in supernatants were examined by enzyme linked immunosorbent assay （ELISA）. The mRNA expressions of VEGF and Ang-2 in cultured HT-29 were determined by real-time quantitative reverse-transcription polymerase chain reaction. Our results showed that treatment of meloxicam of different concentrations and for various lengths of time had a cytotoxicic effect on the cell proliferation of HT-29 cells in a concentration-dependant and time-dependant manner. Cell cycle analysis showed that the cells were mainly blocked in G0/G1 phase. The VEGF and Ang-2 protein levels in supernatants of the culture medium were decreased gradually in a concentration-dependent or time-dependent fashion. The mRNA expression of cox-2, VEGF and Ang-2 showed a gradual and concentration-dependent reduction. It is concluded that meloxicam can reduce the expression of VEGF and Ang-2 at the protein and mRNA level in colon carcinoma cell line.

Apelin and vascular endothelial growth factor are associated with mobilization of endothelial progenitor cells after acute myocardial infarction

This study was designed to determine the levels of early endothelial progenitor cells （EPCs）, apelin, vascu- lar endothelial growth factor （VEGF） and stromal cell-derived growth factor-1 （SDF-1） after acute myocardial infarction （AMI）, and to investigate the relationships between these cytokines and early EPCs. Early EPCs, de- fined as CD133＋, KDR＋, and CD34~ cells, were quantified by flow cytometry. The levels of early EPCs and those cytokines in AMI patients were significantly different from those with coronary artery disease or controls （P 〈 0.05）. Plasma apelin levels were inversely correlated with Gensini score and early EPCs （both P 〈 0.01）. Early EPCs, VEGF and SDF-1 showed different patterns of changes in AMI patients during the first 24 h. The trend in the change of early EPCs was proportionally correlated with that of VEGF （P 〈 0.05）. AMI patients exhibited in- creased early EPCs with remarkably decreased apelin levels and enhanced VEGF levels.

Response of the sensorimotor cortex of cerebral palsy rats receiving transplantation of vascular endothelial growth factor 165-transfected neural stem cells

Neural stem cells are characterized by the ability to differentiate and stably express exogenous ge- nes. Vascular endothelial growth factor plays a role in protecting local blood vessels and neurons of newborn rats with hypoxic-ischemic encephalopathy. Transplantation of vascular endothelial growth factor-transfected neural stem cells may be neuroprotective in rats with cerebral palsy. In this study, 7-day-old Sprague-Dawley rats were divided into five groups： （1） sham operation （control）, （2） cerebral palsy model alone or with （3） phosphate-buffered saline, （4） vascular en- dothelial growth factor 165 ＋ neural stem cells, or （5） neural stem cells alone. The cerebral palsy model was established by ligating the left common carotid artery followed by exposure to hypox- ia. Phosphate-buffered saline, vascular endothelial growth factor ＋ neural stem cells, and neural stem cells alone were administered into the sensorimotor cortex using the stereotaxic instrument and microsyringe. After transplantation, the radial-arm water maze test and holding test were performed. Immunohistochemistry for vascular endothelial growth factor and histology using hematoxylin-eosin were performed on cerebral cortex. Results revealed that the number of vas- cular endothelial growth factor-positive cells in cerebral palsy rats transplanted with vascular endothelial growth factor-transfected neural stem cells was increased, the time for finding water and the finding repetitions were reduced, the holding time was prolonged, and the degree of cell degeneration or necrosis was reduced. These findings indicate that the transplantation of vascu- lar endothelial growth factor-transfected neural stem cells alleviates brain damage and cognitive deficits, and is neuroprotective in neonatal rats with hypoxia ischemic-mediated cerebral palsy.

Early expressions of hypoxia-inducible factor 1alpha and vascular endothelial growth factor increase the neuronal plasticity of activated endogenous neural stem cells after focal cerebral ischemia

Endogenous neural stem cells become ＂activated＂ after neuronal injury, but the activation sequence and fate of endogenous neural stem cells in focal cerebral ischemia model are little known. We evaluated the relationships between neural stem cells and hypoxia-inducible factor-1α and vascular endothelial growth factor expression in a photothromobotic rat stroke model using immunohistochemistry and western blot analysis. We also evaluated the chronological changes of neural stem cells by 5-bromo-2′-deoxyuridine（BrdU） incorporation. Hypoxia-inducible factor-1α expression was initially increased from 1 hour after ischemic injury, followed by vascular endothelial growth factor expression. Hypoxia-inducible factor-1α immunoreactivity was detected in the ipsilateral cortical neurons of the infarct core and peri-infarct area. Vascular endothelial growth factor immunoreactivity was detected in bilateral cortex, but ipsilateral cortex staining intensity and numbers were greater than the contralateral cortex. Vascular endothelial growth factor immunoreactive cells were easily found along the peri-infarct area 12 hours after focal cerebral ischemia. The expression of nestin increased throughout the microvasculature in the ischemic core and the peri-infarct area in all experimental rats after 24 hours of ischemic injury. Nestin immunoreactivity increased in the subventricular zone during 12 hours to 3 days, and prominently increased in the ipsilateral cortex between 3–7 days. Nestin-labeled cells showed dual differentiation with microvessels near the infarct core and reactive astrocytes in the peri-infarct area. BrdU-labeled cells were increased gradually from day 1 in the ipsilateral subventricular zone and cortex, and numerous BrdU-labeled cells were observed in the peri-infarct area and non-lesioned cortex at 3 days. BrdU-labeled cells rather than neurons, were mainly co-labeled with nestin and GFAP. Early expressions of hypoxia-inducible factor-1α and vascular endothelial growth factor after ischemia made up the microenvironment to increase the neuronal plasticity of activated endogenous neural stem cells. Moreover, neural precursor cells after large-scale cortical injury could be recruited from the cortex nearby infarct core and subventricular zone.

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.

Transplantation of vascular endothelial growth factor-modified neural stem/progenitor cells promotes the recovery of neurological function following hypoxic-ischemic brain damage

Neural stem/progenitor cell （NSC） transplantation has been shown to effectively improve neurological function in rats with hypoxic-isch- emic brain damage. Vascular endothelial growth factor （VEGF） is a signaling protein that stimulates angiogenesis and improves neural regeneration. We hypothesized that transplantation of VEGF-transfected NSCs would alleviate hypoxic-ischemic brain damage in neo- natal rats. We produced and transfected a recombinant lentiviral vector containing the VEGF165gene into cultured NSCs. The transfected NSCs were transplanted into the left sensorimotor cortex of rats 3 days after hypoxic-ischemic brain damage. Compared with the NSCs group, VEGF mRNA and protein expression levels were increased in the transgene NSCs group, and learning and memory abilities were significantly improved at 30 days. Furthermore, histopathological changes were alleviated in these animals. Our findings indicate that transplantation of VEGF-transfected NSCs may facilitate the recovery of neurological function, and that its therapeutic effectiveness is better than that of unmodified NSCs.

Lentiviral-mediated vascular endothelial growth factor 165 gene transfer into neural stem cells promotes proliferation

We constructed a lentiviral vector carrying vascular endothelial growth factor 165, which was used to transfect neural stem cells. The transfection rate was approximately 50%, as determined by flow cytometry. Vascular endothelial growth factor protein was detected in neural stem cells and promoted proliferation.

Neuron-specific enolase expression in a rat model of radiation-induced brain injury following vascular endothelial growth factor-modified neural stem cell transplantation

BACKGROUND： Previous studies have shown that transplantation of vascular endothelial growth factor （VEGF）-modified neural stem cells （NSC） provides better outcomes, compared with neural stem cells, in the treatment of brain damage. OBJECTIVE： To compare the effects of VEGF-modified NSC transplantation and NSC transplantation on radiation-induced brain injury, and to determine neuron-specific enolase （NSE） expression in the brain. DESIGN, TIME, AND SETTING： The randomized, controlled study was performed at the Linbaixin Experimental Center, Second Affiliated Hospital, Sun Yat-sen University, China from November 2007 to October 2008. MATERIALS： VEGF-modified C17.2 NSCs were supplied by Harvard Medical School, USA. Streptavidin-biotin-peroxidase-complex kit （Boster, China） and 5, 6-carboxyfluorescein diacetate succinimidyl ester （Fluka, USA） were used in this study. METHODS： A total of 84 Sprague Dawley rats were randomly assigned to a blank control group （n = 20）, model group （n = 20）, NSC group （n = 20）, and a VEGF-modified NSC group （n = 24）. Rat models of radiation-induced brain injury were established in the model, NSC, and VEGF-modified NSC groups. At 1 week following model induction, 10 pL （5 ×10^4 cells/μL） VEGF-modified NSCs or NSCs were respectively infused into the striatum and cerebral cortex of rats from the VEGF-modified NSC and NSC groups. A total of 10μL saline was injected into rats from the blank control and model groups. MAIN OUTCOME MEASURES： NSE expression in the brain was detected by immunohistochemistry following VEGF-modified NSC transplantation. RESULTS： NSE expression was significantly decreased in the brains of radiation-induced brain injury rats （P 〈 0.05）. The number of NSE-positive neurons significantly increased in the NSC and VEGF-modified NSC groups, compared with the model group （P 〈 0.05）. NSE expression significantly increased in the VEGF-modified NSC group, compared with the NSC group, at 6 weeks following transplantation （P 〈 0.05）. CONCLUSION： VEGF-modified NSC transplantation increased NSE expression in rats with radiation-induced brain injury, and the outcomes were superior to NSC transplantation.

A CORRELATIVE STUDY OF Ki67 AND VASCULAR ENDOTHELIAL GROWTH FACTOR AND THEIR VALUE IN LARYNGEAL SQUAMOUS CELL CARCINOMA

Objective： To study the correlation between Ki67 and vascular endothelial growth factor（VEGF） and the significance of their expression in laryngeal squamous cell carcinoma（LSCC）. Methods： The expressions of Ki67 and VEGF in 40 cases of LSCC and 5 cases of normal laryngeal mucosa were examined by immunohistochemical staining. Results： The expression levels of Ki67 and VEGF in LSCC tissue were higher than in normal laryngeal mucosa （Ki67： P〈0.001, VEGF： P〈0.001）. The two indexes＇ levels in patients of different age or different sex had no significant difference （P〉0.05）. They were higher in LSCC with metastasis of lymph nodes than in patients without metastasis （Ki67： P=0.034, VEGF： P=0.006）. The expressions of the two genes elevated correspondingly along with the development of LSCC T stage （P〈0.05）. In addition, correlation analysis indicated that the expression of Ki67 had a positive correlation with VEGF in LSCC（r=0.823, P〈0.01）. Conclusion： Ki67 and VEGF are objective indexes for the biological behavior of LSCC, and they might be helpful to the diagnosis, treatment and prognosis of laryngocarcinoma.

EXPRESSION OF VASCULAR ENDOTHELIAL GROWTH FACTOR AND BASIC FIBROBLAST GROWTH FACTOR IN HUMAN NON-SMALL CELL LUNG CANCER AND THEIR CLINICAL SIGNIFICANCE

Objective: To explore the expression of vascular endothelial growth factor(VEGF) and basic fibroblast growth factor(bFGF) in non-small cell lung cancer(NSCLC) and theIR clinical significance. Methods: The expression of VEGF and bFGF was examined at the protein levels by immunohistochemical staining in 96 NSCLC patients, and in 36 of which at the mRNA levels by reverse transcription-PCR analysis. Results: VEGF mRNAs were expressed predominately as its secretory forms (VEGF121 and VEGF165) in NSCLC tissues. The positive ratios of VEGF121 and VEGF165 were 69.5%(25 of 36) and 41.7%(15 of 36) respectively. The positive ratio of bFGF was 52.8(19 of 36) in the same tumor specimens. The positive ratios of VEGF and bFGF at protein levels were 55.55%(20 of 36) and 58.33%(21 of 36) respectively. A significant positive correlation was observed between VEGF and bFGF expression in NSCLC tissues(P=0.002). No significant interrelationship between VEGF, bFGF expression and clinical data(age, sex, histological subtype differentiation, P-stage, metastasis and survival) was found. Conclusions: VEGF and bFGF may play an important role in angiogenesis and act in a synergistic manner in NSCLC.

The effect of Chinese herbal medicine“heche assisted pregnancy recipe”on endometrial estrogen and progesterone receptor,proliferating cell nuclear antigen and vascular endothelial growth factor in the patients with infertility

Objectives:To investigate the effect of Chinese herbal medicine"heche assisted preg-nancy recipe (HCAPR)" on estrogen receptor(ER), progesterone receptor (PR), pro-lifierating cell nuclear antigen(PCNA) and vascular endothelial growth factor (VEGF)in endometrium of infertile women.Methods: The S-P immunohistochemical assay was used to observe expression ofER, PR , PCNA and VEGF in late proliferative phase before and after the HCAPR treat-ment.Results: After the treatment, the expression of ER,PR,PCNA and VEGF in nucleiof glandular epithelium and stromal cells was significantly stronger (all P＜0. 001) re-spectively than that before treatment , especially the expression of PCNA and VEGF.Conclusions: These results suggest that traditional Chinese medicine HCAPR oftonifying kidney and regulating menstruation increased the synthesis of ER,PR, PCNAand VEGF, which may promote normal growth and development of the endometrium ,improve the micro-environment of the endometrium, and enhance uterine receptivity.The evidence may provide theoretical basis for therapy infertility with Chinese herbalmedicine.

Rhizoma Chuanxiong regulates vascular endothelial growth factor production in hypoxic human umbilical vein endothelial cells in vitro and in peri-infarct rat brain tissue

BACKGROUND： Vascular endothelial growth factor （VEGF） acts as ＂molecular bridge＂ following ischemic stroke to improve and restore blood supply and reduce infarction volume. Clinical studies have demonstrated the efficacy of Rhizoma Chuanxiong （Chuanxiong） in the treatment of ischemic cerebrovascular diseases. However, whether it promotes endogenous VEGF expression in ischemic stroke remains unknown. OBJECTIVE： To investigate the influence of Rhizoma Chuanxiong on VEGF production in vitro cultured human umbilical vein endothelial cells and on VEGF expression in ischemic cerebral tissues to explore its role in angiogenesis. DESIGN, TIME AND SETTING： In vitro basic comparison of traditional Chinese drug-containing serum pharmacology; in vivo randomized, controlled, animal experiment. This study was performed at the Medical Laboratory of West China Hospital, Sichuan University between December 2002 and April 2004. MATERIALS： Two Chinese rabbits were selected. One was intragastrically perfused with 5.8 g/kg Rhizoma Chuanxiong extract twice per day for three consecutive days to prepare Rhizoma Chuanxiong extract-containing serum. The remaining rabbit was intragastrically perfused with the same volume of normal saline twice per day for three consecutive days. Rhizoma Chuanxiong extract was provided by Beijing Traditional Chinese Medicine Research Institute, predominantly composed of ligustrazine, ligustilide, and ferulic acid. ChemiKineTM human VEGF Kit was purchased from Chemicon, USA; mouse anti-VEGF monoclonal antibody and biotin-goat anti-mouse IgG were purchased from Santa Cruz Biotechnology. Inc., USA. METHODS： （1） In vitro experiment： in vitro cultured human umbilical vein endothelial cells were separately incubated in rabbit serum with 10% Rhizoma Chuanxiong extract, normal medium without rabbit serum, and rabbit serum without Rhizoma Chuanxiong extract （blank control）. In addition, cells from the three groups were incubated under normoxia （5% CO2, 95% air） and hypoxia （1% 02, 5% CO2, 94% N2）, respectively, for 24 hours. （2） In vivo experiment： a total of 4/44 Sprague Dawley rats were selected for the sham-operated group （no occlusion）, and the remaining rats were used to establish a cerebral ischemiaJreperfusion model by suture occlusion. 32 animals with ischemia/reperfusion injury were randomly divided into treatment and model groups, with 16 rats in each group. Both groups were intraperitoneally infused with 0.58 g/kg Rhizoma Chuanxiong extract and normal saline two hours following reperfusion. The sham-operated group was administrated normal saline. Animals were treated with saline or Chuanxiong extracts （0.58 g/kg） twice per day for three consecutive days. MAIN OUTCOME MEASURES： In vitro experiment： VEGF concentration was detected in each group by enzyme-linked immunosorbent assay. In vivo experiment： behavioral alterations of rats were evaluated by neurological function scale; infarct volume was assessed by hematoxylin-eosin staining; VEGF protein expression in the infarct regions was determined by immunohistochemistry. RESULTS： （1） VEGF levels were similar between the three groups under normexic condition （P 〉 0.05）; while hypoxia induced VEGF production （P 〈 0.01 ）. VEGF levels in the drug-containing serum group were particularly higher compared with the other groups （P 〈 0.01）. （2） Compared with normal saline treatment, Rhizoma Chuanxiong extract significantly improved the neurological scale and reduced cerebral infarct volumes （P〈 0.05）. The percent of VEGF-positive cells was significantly greater than the model group （P 〈 0.05）. The sham-operated group exhibited normal neurological function, with no infarct focus. CONCLUSION： Rhizoma Chuanxiong extract-containing rabbit serum effectively promoted cultured VEGF production under hypoxia. Rhizoma Chuanxiong extract upregulated VEGF expression in the infarct region, improved neurological function, and reduced infarct size.

Vascular endothelial growth factor and angiopoietins regulate sinusoidal regeneration and remodeling after partial hepatectomy in rats

AIM： To study the regulatory mechanisms of sinusoida regeneration after partial hepatectomy. METHODS： We invesldgated the expression of angiopoietin （Ang）-1, Ang-2, Tie-2, and vascular endothelial growth factor （VEGF） in regenerating liver tissue by quantitative reverse-transcription polymerase chain reaction （RT- PCR） using a LightCycler （Roche Diagnostics） and also immunohistochemical staining after 70% hepatectomy in rats. In the next step, we isolated liver cells （hepatocytes, sinusoidal endothelial cell （SEC）, Kupffer cell, and hepatic stellate cells （HSC）） from regenerating liver tissue by in situ collagenase perfusion and counterflow elutriation, to determine potential cellular sources of these angiogenic factors after hepatectomy. Proliferation and apoptosis of SECs were also evaluated by proliferating cell nuclear antigen （PCNA） staining and the terminal deoxynucleotidyl transferase d-uridine triphosphate nick end labeling （TUNEL） assay, respectively. RESULTS： VEGF mRNA expression increased with a peak at 72 h after hepatectomy, decreasing thereafter. The expression of Ang-1 mRNA was present at detectable levels before hepatectomy and increased slowly with a peak at 96 h. Meanwhile, Ang-2 mRNA was hardly detected before hepatectomy, but was remarkably induced at 120 and 144 h. In isolated cells, VEGF mRNA expression was found mainly in the hepatocyte fraction. Meanwhile, mRNA for Ang-1 and Ang-2 was found in the SEC and HSC fractions, but was more prominent in the latter. The PCNA labeling index of SECs increased slowly, reaching a peak at 72 h, whereas apoptotic SECs were detected between 120 h and 144 h. CONCLUSION： Ang-Tie system, together with VEGF, plays a critical role in regulating balance between SEC proliferation and apoptosis during sinusoidal regeneration after hepatectomy. However, the VEGF system plays a more important role in the early phase of sinusoidal regeneration than angiopoietin/Tie system.

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.